Masahumi Kawaguchi

Single-neuron diversity generated by Protocadherin-β cluster in mouse central and peripheral nervous systems

The generation of complex neural circuits depends on the correct wiring of neurons with diverse individual characteristics. To understand the complexity of the nervous system, the molecular mechanisms for specifying the identity and diversity of individual neurons must be elucidated. The clustered protocadherins (Pcdh) in mammals consist of approximately 50 Pcdh genes (Pcdh-α, Pcdh-β, and Pcdh-γ) that encode cadherin-family cell surface adhesion proteins. Individual neurons express a random combination of Pcdh-α and Pcdh-γ, whereas the expression patterns for the Pcdh-β genes, 22 one-exon genes in mouse, are not fully understood. Here we show that the Pcdh-β genes are expressed in a 3′-polyadenylated form in mouse brain. In situ hybridization using a pan-Pcdh-β probe against a conserved Pcdh-β sequence showed widespread labeling in the brain, with prominent signals in the olfactory bulb, hippocampus, and cerebellum. In situ hybridization with specific probes for individual Pcdh-β genes showed their expression to be scattered in Purkinje cells from P10 to P150. The scattered expression patterns were confirmed by performing a newly developed single-cell 3′-RACE analysis of Purkinje cells, which clearly demonstrated that the Pcdh-β genes are expressed monoallelically and combinatorially in individual Purkinje cells. Scattered expression patterns of individual Pcdh-β genes were also observed in pyramidal neurons in the hippocampus and cerebral cortex, neurons in the trigeminal and dorsal root ganglion, GABAergic interneurons, and cholinergic neurons. Our results extend previous observations of diversity at the single-neuron level generated by Pcdh expression and suggest that the Pcdh-β cluster genes contribute to specifying the identity and diversity of individual neurons.

Reptiles: a new model for brain evo-devo research.

Vertebrate brains exhibit vast amounts of anatomical diversity. In particular, the elaborate and complex nervous system of amniotes is correlated with the size of their behavioral repertoire. However, the evolutionary mechanisms underlying species-specific brain morphogenesis remain elusive. In this review we introduce reptiles as a new model organism for understanding brain evolution. These animal groups inherited ancestral traits of brain architectures. We will describe several unique aspects of the reptilian nervous system with a special focus on the telencephalon, and discuss the genetic mechanisms underlying reptile-specific brain morphology. The establishment of experimental evo-devo approaches to studying reptiles will help to shed light on the origin of the amniote brains.

Myelination triggers local loss of axonal CNR/protocadherinα family protein expression

The cadherin‐related neuronal receptor (CNR)/protocadherin (Pcdh) α family is one of the diverse protocadherin families expressed in developing axons. We observed a strong axonal expression of these proteins at late embryonic and early postnatal stages corresponding to regions where fibers had not yet been myelinated. We therefore followed the postnatal localization of CNR/Pcdhα protein in major axonal tracts, such as the internal capsule, lateral olfactory tract, and optic nerve, and found that its axonal localization was dramatically lost in parallel with the increased expression of myelin markers. Moreover, the hypomyelinated optic nerve tracts of the myelin‐deficient Shiverer mouse exhibited elevated levels of CNR/Pcdhα expression. These axonal expression patterns of CNR/Pcdhα in wild‐type and Shiverer mice were similar to those of growth associated protein 43 (GAP‐43) and L1, both of which are associated with axonal maturation. Thus, myelination may be a trigger for the local loss of axonal CNR/Pcdhα protein, and this process may be important in the maturation of neural circuits.

Effect of heavy oil on the development of the nervous system of floating and sinking teleost eggs.

Heavy oil (HO) on the sea surface penetrates into fish eggs and prevents the normal morphogenesis. To identify the toxicological effects of HO in the context of the egg types, we performed exposure experiments using floating eggs and sinking eggs. In the course of development, HO-exposed embryos of floating eggs showed abnormal morphology, whereas early larva of the sinking eggs had almost normal morphology. However, the developing peripheral nervous system of sinking eggs showed abnormal projections. These findings suggest that HO exposed fishes have problems in the developing neurons, although they have no morphological malformations. Through these observations, we conclude that HO is strongly toxic to floating eggs in the morphogenesis, and also affect the neuron development in both floating and sinking eggs.

Expression levels of Protocadherin-α transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions

The mouse protocadherin (Pcdh) clusters, Pcdh-alpha, -beta, and -gamma, are located on chromosome 18. Many polymorphic variations are found in the Pcdh-alpha genes in wild-derived and laboratory mouse strains. In comparing the expression levels of Pcdh-alpha isoforms among several strains, we observed lower expression levels of Pcdh-alpha9 in BLG2 and BFM/2, and of Pcdh-alpha8 in C57BL/6 (B6) than in the other strains. For Pcdh-alpha8, high DNA methylation (72.7%) in the promoter region was found only in B6, whereas 36.4-44.3% methylation was seen in the other strains. On the other hand, the Pcdh-alpha9 DNA-methylation levels were similar (23.6-36.3%) among the strains regardless of the difference in expression levels. Interestingly, however, the Pcdh-alpha9 variable exon in both BLG2 and BFM/2 included a premature termination codon (PTC) generated by a nucleotide deletion or insertion. Treatment with emetine, a potent inhibitor of nonsense-mediated mRNA decay (NMD), increased the expression level of Pcdh-alpha9 from the BLG2-Pcdh-alpha locus. These data indicate that the transcription levels of mature Pcdh-alpha mRNAs are decreased by the DNA-methylation state of the Pcdh-alpha promoter regions and by the NMD pathway during RNA maturation. And we correct some previous data on Sugino, H., Toyama, T., Taguchi, Y., Esumi, S., Miyazaki, M., Yagi, T., (2004) Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines, Gene 337 91-103.

Evolutionary divergence of trigeminal nerve somatotopy in amniotes.

The trigeminal circuit relays somatosensory input from the face into the central nervous system. In central nuclei, the spatial arrangement of neurons reproduces the physical distribution of peripheral receptors, thus generating a somatotopic facial map during development. In mice, the ophthalmic, maxillary, and mandibular trigeminal nerve branches maintain a somatotopic segregation and generate spatially organized patterns of connectivity within hindbrain target nuclei. To investigate conservation of somatotopic organization, we compared trigeminal nerve organization in turtle, chick, and mouse embryos. We found that, in the turtle, mandibular and maxillary ganglion neuron rostrocaudal segregation and trigeminal tract somatotopy are similar to mouse. In contrast, chick mandibular ganglion neurons are located rostrally to maxillary neurons, with some intermingling, supporting previous observations (Noden [1980], J Comp Neurol 190:429–444). This organization results in an inversion of the relative positions and less precise axonal sorting of the maxillary and mandibular branches within the trigeminal tract, as compared to mouse and turtle. Moreover, using the turtle and chick orthologs of Drg11 in combination with Hoxa2 expression and axonal tracings from the periphery, we mapped the chick PrV nucleus position to rhombomere 1, confirming previous studies (Marin and Puelles [1995], Eur J Neurosci 7:1714–1738) and in contrast to mouse PrV, which mainly maps to rhombomere 2–3 (Oury et al. [2006], Science 313:1408–1413). Thus, somatotopy of trigeminal ganglion and nerve organization is only partially conserved through amniote evolution, possibly in relation to the modification of facial somatosensory structures and morphologies. J. Comp. Neurol. 1378–1394, 2013.

Development of the dorsal ramus of the spinal nerve in the mouse embryo: Involvement of semaphorin 3A in dorsal muscle innervation

The spinal nerve, which is composed of dorsal root ganglion (DRG) sensory axons and spinal motor axons, forms the dorsal ramus projecting to the dorsal musculature. By using the free‐floating immunohistochemistry method, we closely examined the spatiotemporal pattern of the formation of the dorsal ramus and the relationship between its projection to the myotome/dorsal musculature and semaphorin 3A (Sema3A), which is an axonal guidance molecule. In embryonic day (E) 10.5–E11.5 wild‐type mouse embryos, we clearly showed the existence of a waiting period for the dorsal ramus projection to the myotome. In contrast, in E10.5–E11.5 Sema3A‐deficient embryos, the dorsal ramus fibers projected beyond the edge of the myotome without exhibiting the waiting period for projection. These results strongly suggest that the delayed innervation by dorsal ramus fibers may be caused by Sema3A‐induced axon repulsion derived from the myotome. Next, by performing culture experiments, we confirmed that E12.5 mouse axons responded to Sema3A‐induced repulsion. Together, our results imply that Sema3A may play a key role in the proper development of the dorsal ramus projection.

Disruption of Sema3A expression causes abnormal neural projection in heavy oil exposed Japanese flounder larvae.

It has been well known that oil spills cause serious problems in the aquatic organisms. In particular, some species of teleosts, which develop on the sea surface thought to be affected by heavy oil (HO). During the embryogenesis, the nervous system is constructed. Therefore, it is important to study the toxicological effects of HO on the developing neurons. We exposed HO to eggs of Japanese flounder (Paralichthys olivaceus) and investigated the neural disorder. In larvae exposed by HO at the concentration of 8.75 mg/L, the facial and lateral line nerves partially entered into the incorrect region and the bundle was defasciculated. Furthermore, in the HO-exposed larvae, Sema3A, a kind of axon guidance molecule, was broadly expressed in second pharyngeal arch, a target region of facial nerve. Taken together, we suggested the possibility that the abnormal expression of Sema3A affected by HO exposure causes disruption of facial nerve scaffolding.

Pyrene induces a reduction in midbrain size and abnormal swimming behavior in early-hatched pufferfish larvae.

Spills of heavy oil (HO) have an adverse effect on marine life. We have demonstrated previously that exposure to HO by fertilized eggs of the pufferfish (Takifugu rubripes) induces neural disruption and behavioral abnormality in early-hatched larvae. Here, two kinds of polycyclic aromatic hydrocarbons, pyrene and phenanthrene, were selected to examine their toxic effects on larval behavior of another pufferfish species (T. niphobles). Larvae exposed to pyrene or phenanthrene exhibited no abnormalities in morphology. However, those exposed to pyrene but not phenanthrene swam in an uncoordinated manner, although their swimming distance and speed were normal. The optic tectum, a part of the midbrain, of pyrene-exposed larvae did not grow to full size. Thus, these findings are indicated that pyrene might be a contributor to the behavioral and neuro-developmental toxicity, although there is no indication that it is the only compound participating in the toxicity of the heavy oil mixture.

Relationship between DNA methylation and gene expression in the protocadherin gene cluster

P3-c04 G protein-coupled receptor (GPCR) signaling induces activity-dependent gene expression through the modulation of N-methyl-d-aspartate receptor (NMDA-R) in neurons Masaaki Tsuda 1 , Mamoru Fukuchi 1, Shinjiro Watanabe 1, Yuki Kuwana 1, Ichiro Takasaki 2, Akiko Tabuchi 1 1 Department Biol. Chem., Grad. Sch. of Med. and Pharm. Sci., University of Toyama, Toyama 2 Div. of Mol. Gen. Res., Life. Sci. Res. Ctr., University of Toyama, Toyama