尾身 実

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered thatEphA7andEfnA5are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically,EphA7andEfnA5forward and reverse inhibitory signals direct collateral extension such thatEphA7-positive frontal and occipital cortical areas extend their axon collaterals into theEfnA5-negative rostral part of the basilar pons, whereasEfnA5-positive parietal cortical areas extend their collaterals into theEphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets. SIGNIFICANCE STATEMENTOur findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization—both within each brain region and between connected brain regions—may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Abstract Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513G>A mutation. The patient’s dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513G>A heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.

To elucidate a gene function, in vivo analysis is indispensable. We can carry out gain and loss of function experiment of a gene of interest by electroporation in ovo and ex ovo culture system on early-stage and advanced-stage chick embryos, respectively. In this section, we introduce in/ex ovo electroporation methods for the development of the chick central nervous system and visual system investigation.

It has been shown that strong Fgf8 signal activates Ras-ERK signaling pathway to determine metencephalon, which consists of rhombomere 1 (r1), where the cerebellum differentiates, and isthmus (r0). The present study was undertaken to check if Ets type transcription factor Pea3 functions downstream of Ras-ERK signaling to determine metencephalon. Pea3 misexpression resulted in repression of Otx2 expression in the mesencephalon, induction of Gbx2 and Fgf8 expression in the mesencephalon, and differentiation of the trochlear neurons in the posterior mesencephalon. Fate change of the tectum to the cerebellum did not occur. Repression of Pea3 function by misexpressing the chimeric molecule of Engrailed repressor domain EH1 and Pea3 (eh1-Pea3) resulted in induction of Otx2 expression in the metencephalon, repression of Gbx2 and Fgf8 expression in the metencephalon, and differentiation of the oculomotor neurons in the isthmus. It was concluded that Pea3 plays a pivotal role in determination of the isthmus (r0) property downstream of Fgf8-Ras-ERK signaling.

Cell positioning and neuronal network formation are crucial for proper brain function. Disrupted-in-Schizophrenia 1 (DISC1) is anterogradely transported to the neurite tips, together with Lis1, and functions in neurite extension via suppression of GSK3 beta activity. Then, transported Lis1 is retrogradely transported and functions in cell migration. Here, we show that DISC1-binding zinc finger protein (DBZ), together with DISC1, regulates mouse cortical cell positioning and neurite development in vivo. DBZ hindered Ndel1 phosphorylation at threonine 219 and serine 251. DBZ depletion or expression of a double-phosphorylated mimetic form of Ndel1 impaired the transport of Lis1 and DISC1 to the neurite tips and hampered microtubule elongation. Moreover, application of DISC1 or a GSK3 beta inhibitor rescued the impairments caused by DBZ insufficiency or double-phosphorylated Ndel1 expression. We concluded that DBZ controls cell positioning and neurite development by interfering with Ndel1 from disproportionate phosphorylation, which is critical for appropriate anterograde transport of the DISC1-complex.

The optic tectum is a visual center of nonmammalian vertebrates derived from the mesencephalon. In this review, function of Engrailed (En) in tectum development is reviewed. En plays crucial roles at three steps of tectum development. First, Engrailed is expressed in the mesencephalon and the metencephalon and essential for the regionalization of the mesencephalon. En is expressed in a gradient of caudal-to-rostral in the tectum primordial, and regulates the rostrocaudal polarity of the tectum. In the advanced stage of tectum development, En is expressed in a lamina-specific manner and it is suggested that En regulates cell migration in the tectal laminar formation.

The chick optic tectum consists of 16 laminae. Here, we report contribution of En2 to laminar formation in chick optic tecta. En2 is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. Our results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation.

The optic tectum is a visual center of nonmammalian vertebrates that receives retinal fibers in a retinotopic manner. It has been accepted that retinal fibers project to some superficial laminae of the tectum, but do not go through lamina g of stratum griseum et fibrosum superficiale (SGFS). By a novel fiber-tracing method, we found a novel pathway of retinal fibers that run through deep laminae of the tectum. The retinal fibers that would run through the newly identified pathway first run caudally along the medial edge after invading the tectum, turn laterally, and extend toward the lateral side through the deep pathway. The deep pathway runs through stratum album centrale and stratum fibrosum periventriculare. The fibers that run through the deep pathway do not enter the stratum opticum, where the conventional retinal fibers run. As development proceeds, these fibers decrease and disappear by the adult stage. By the new method, we found that some of the conventional retinal fibers transiently run through lamina g of SGFS and invade laminae h/i. In conclusion, we found distinct but transient retinal fiber pathway in the deep tectal laminae, which have not been thought to be retinorecipient. J. Comp. Neurol. 519:2615-2621, 2011. (C) 2011 Wiley-Liss, Inc.

In vivo electroporation has served as an effective tool for the study of developmental biology. Here we report tetracycline inducible gene knockdown by electroporation. Our system consists of genome integration of a cassette encoding long double strand RNA (dsRNA) of a gene of interest by electroporation, transcription of which is assured by RNA polymerase II, and induction of transcription of dsRNA by tetracyclin. Long dsRNA decapped by ribozyme in the cassette and without poly A tail is processed into siRNA within nuclei. We could successfully induce knockdown of En2 and Coactosin by Dox administration.

In the developing chick leg bud, massive programmed cell death occurs in the interdigital region. Previously, we reported the inhibition of cell death by separation of the interdigital region from neighboring digit cartilage. In this study, we examined the relationship between cell death and cartilaginous tissue in vitro. First, cell fate was observed with Dil that was used to examine cell movement in the distal tip of leg bud. Labeled cells in the prospective digital region were distributed only in the distal region as a narrow band, while cells in the prospective interdigital region expanded widely in the interdigit. In coculture of monolayer cells and a cell pellet tending to differentiate into cartilage, monolayer cells migrated into the cell pellet. These results suggested that digit cartilage tends to recruit neighboring cells into the cartilage during limb development. Next, we observed the relationship between cell death and chondrogenesis in monolayer culture. Apoptotic cell death that could be detected by TUNEL occurred in regions between cartilaginous nodules in mesenchymal cell culture. More apoptotic cell death was detected in the cell culture of leg bud mesenchyme of stage 25/26 than that of leg bud mesenchyme of stage 22 or that of stage 28. The most developed cartilaginous nodules were observed in the cell culture of stage 25/26. Finally, we observed Smp expression in vitro and in vivo. Bmp-2, Bmp-4 and Bmp-7 were detected around the cartilage nodules. When the interdigit was separated from neighboring digit cartilage, Bmp-4 expression disappeared near the cut region but remained near the digit cartilage. This correlation between cell death and cartilaginous region suggests that cartilage tissue can induce apoptotic cell death in the developing chick limb bud due to cell migration accompanying chondrogenesis and Bmp expression.

A fundamental process in limb bud development is the formation of position-dependent cartilage pattern. Cells of the distal mesenchyme maintain positional values as the expression pattern of transcription factors, for example, hox genes, which induce position-related cell differentiation and cell surface differences. Cultured, dissociated limb bud mesenchymal cells segregate from each other, and eventually form cartilage nodules. This sorting out is position-dependent, not cell-type dependent, suggesting that the positional values may be involved. Positional valves were found to be retained in limb bud recombinants. In the chick system, the expression of HoxA13 and HoxD12 was present in the distal half of stage 20 recombinants, whereas these markers were expressed throughout the stages 25 recombinants. In the Xenopus system, multiple digit formation was introduced in limb recombinants, and a position-related relationship between regeneration potency and the multiple digit formation could be established. This determination of multiple digit formation with different stages of limb mesenchyme may be useful in understanding mechanisms of the loss of vertebrate limb regeneration potency. Copyright ©1998 by The Wound Healing Society.