Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

Effects of flurbiprofen on the functional regulation of serotonin transporter and its misfolded mutant.

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

TEAD1 trapping by the Q353R-Lamin A/C causes dilated cardiomyopathy.

Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R-LMNA-related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA-related DCM.

Dynamic changes in the gene expression of zebrafish Reelin receptors during embryogenesis and hatching period.

The brain morphology of vertebrates exhibits huge evolutionary diversity, but one of the shared morphological features unique to vertebrate brain is laminar organization of neurons. Because the Reelin signal plays important roles in the development of the laminar structures in mammalian brain, investigation of Reelin signal in lower vertebrates will give some insights into evolution of vertebrate brain morphogenesis. Although zebrafish homologues of Reelin, the ligand, and Dab1, a cytoplasmic component of the signaling pathway, have been reported, the Reelin receptor molecules of zebrafish are not reported yet. Here, we sought cDNA sequence of zebrafish homologue of the receptors, vldlr and apoer2, and examined their expression patterns by in situ hybridization. Developmental gene expression pattern of reelin, dab1, vldlr, and apoer2 in the central nervous system of zebrafish was compared, and their remarkable expression was detected in the developing laminar structures, such as the tectum and the cerebellum, and also non-laminated structures, such as the pallium. The Reelin receptors exhibited different spatial and temporal gene expression. These results suggest a possibility that duplication and subsequent functional diversity of Reelin receptors contributed to the morphological and functional evolution of vertebrate brain.

Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 -tubulin complex.

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

From meter-long structures that allow nerve cells to stretch across a body to miniscule 'hairs' required for lung cells to clear mucus, many life processes rely on cells sporting projections which have the right size for their role. Networks of hollow filaments known as microtubules shape these structures and ensure that they have the appropriate dimensions. Controlling the length of microtubules is therefore essential for organisms, yet how this process takes place is still not fully elucidated. Previous research has shown that microtubules continue to grow when their end is straight but stop when it is curved. A family of molecular motors known as kinesin-4 participate in this process, but the exact mechanisms at play remain unclear. To investigate, Tuguchi, Nakano, Imasaki et al. focused on the KLP-12 protein, a kinesin-4 equivalent which helps to controls the length of microtubules in the tiny worm Caenorhabditis elegans. They performed genetic manipulations and imaged the interactions between KLP-12 and the growing end of a microtubule using X-ray crystallography. This revealed that KLP-12 controls the length of neurons by inhibiting microtubule growth. It does so by modulating the curvature of the growing end of the filament to suppress its extension. A 'snapshot' of KLP-12 binding to a microtubule at the resolution of the atom revealed exactly how the protein helps to bend the end of the filament to prevent it from growing further. These results will help to understand how nerve cells are shaped. This may also provide insights into the molecular mechanisms for various neurodegenerative disorders caused by problems with the human equivalents of KLP-12, potentially leading to new therapies.

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

GABA modulates development of cerebellar Purkinje cell dendrites under control of endocannabinoid signaling.

Purkinje cells (PCs) are the sole projection neurons in the cerebellar cortex with highly arborized dendrites, on which they receive glutamatergic and GABAergic inputs. Whereas influences of glutamatergic inputs on dendritic development of PCs have been well studied, those of GABA remain elusive. Here we examined effects of GABAergic signaling on dendritogenesis of PCs in dissociated cerebellar cultures. Treatment with GABA(A) agonists such as muscimol altered Purkinje dendrites to longer and less branched morphology, while GABA(A) antagonists resulted in shorter dendrites. In contrast, neither a GABA(B) agonist nor antagonist had major effects on dendritic morphology. Simultaneous addition of a glutamatergic antagonist cocktail or the Trk receptor antagonist K252a did not block muscimol. Furthermore, blockade of endocannabinoid signaling by either AM251 or tetrahydrolipstatin resulted in longer and less branched dendrites similar to those treated with GABA(A) agonists suggesting upstream regulation by endocannabinoids. Notably, whereas Purkinje dendrites extended in random directions in the presence of muscimol, they oriented to coexisting GABAergic interneurons when treated with AM251. Taken together, our results postulate the hypothesis that GABA released from the cerebellar interneurons modulates dendritogenesis of PCs in an endocannabinoid-dependent manner in the developing cerebellar cortex.

PKH26 is an excellent retrograde and anterograde fluorescent tracer characterized by a small injection site and strong fluorescence emission.

The fluorescent dye PKH26, which binds mainly to the cell membrane, has long stability that enables the tracing of PKH26-labeled transplanted cells in host tissue. In the present study, we examined whether this fluorescent dye works as a retrograde or anterograde tracer to label neural networks within the central nervous system of adult and postnatal day 3 (P3) mice. A small injection of the dye into the medullospinal junction resulted in the retrograde labeling of corticospinal tract (CST) neurons in layer V of the sensory-motor cortex both in the adult mice and pups. Injection of the dye into the motor cortex of the P3 pups resulted in the anterograde labeling of CST fibers at a single fiber resolution level, although a similar injection of the dye into the motor cortex of the adult mice failed to stain CST fibers anterogradely. These results suggest that, while PKH26 works as a retrograde or anterograde tracer, anterograde labeling of the adult tracts can not be expected.

Constant existence of the sensory branch of the nerve to the pyramidalis distributing to the upper margin of the pubic ramus.

Twenty-one sides of 11 adult Japanese cadavers were investigated, and 2 of 21 sides exhibited absence of the pyramidalis. We observed that all of the nerves to the pyramidalis included the sensory nerve branch, which distributed to the aponeurotic tissue in the upper area of the pubic ramus. To investigate the clinical relevance and developmental process of the pyramidalis, detailed innervation patterns of the pyramidalis and the lumber plexus were examined and compared with the case of absent pyramidalis. The nerves to the pyramidalis could be classified into five types by the derivative nerves and two subtypes by their courses associated with the funiculus spermaticus. In the cases of absent pyramidalis, similar sensory branches distributed close to the upper area of the pubic ramus. We deduced that the sensory branch extended along with the muscular branch to the pyramidalis after development of the pyramidalis and that only the sensory branch remained in cases in which the pyramidalis disappeared. The two subtypes might associate with descensus testis. Surgeons performing inguinal hernia repair using a mesh and tension-free surgical technique should preserve the nerves around the funiculus spermaticus to avoid diminished proprioception in the lower abdominal wall.

The zebrafish pob gene encodes a novel protein required for survival of red cone photoreceptor cells.

The zebrafish mutant, partial optokinetic response b (pob), was isolated using an N-ethyl N-nitrosourea (ENU)-based screening strategy designed to identify larvae with defective optokinetic responses in red but not white light. Previous studies showed that red-light blindness in pob is due to the specific loss of long-wavelength photoreceptor cells via an apoptotic mechanism. Here, we used positional cloning to identify the mutated pob gene. We find that pob encodes a highly conserved 30-kDa protein of unknown function. To demonstrate that the correct gene was isolated, we used the Tol2 transposon system to generate transgenic animals and rescue the mutant phenotype. The Pob protein contains putative transmembrane regions and protein-sorting signals. It is localized to the inner segment and synapse in photoreceptor cells, and when expressed in COS-7 cells it localizes to intracellular compartments. We also show that the degeneration of red cone photoreceptors in the mutants occurs independently of light. On the basis of our findings, we propose that Pob is not involved in phototransduction but rather plays an essential role in protein sorting and/or trafficking.

Apolipoprotein E and Reelin ligands modulate tau phosphorylation through an apolipoprotein E receptor/disabled-1/glycogen synthase kinase-3beta cascade.

Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimer's disease pathology. Mice lacking Reelin (Reln), double-knockouts lacking the VLDL receptor (VLDLR) and ApoE receptor2 (ApoER2), and mice lacking disabled-1 (Dab1) display increased levels of phosphorylated tau. Because Reln binds to recombinant ApoE receptors, assembly of a Reln/ApoE-receptor/Dab1 (RAD) complex may initiate a signal transduction cascade that controls tau phosphorylation. Conversely, disruption of this RAD complex may increase tau phosphorylation and lead to neurodegeneration. To substantiate this concept, we mated Reln-deficient mice to ApoE-deficient mice and found that in the absence of Reln, tau phosphorylation increased as the amount of ApoE decreased. Paralleling the change in tau phosphorylation levels, we found that GSK-3beta activity increased in Reln-deficient mice and further increased in mice lacking both Reln and ApoE. CDK-5 activity was similar in mice lacking Reln, ApoE, or both. GSK-3beta and CDK-5 activity increased in Dab1-deficient mice, independent of ApoE levels. Further supporting the idea that increased tau phosphorylation results primarily from increased kinase activity, the activity of two phosphatases was similar in all conditions tested. These data support a novel, ligand-mediated signal transduction cascade--initiated by the assembly of a RAD complex that suppresses kinase activity and controls tau phosphorylation.

Postnatal Development of the Corticospinal Tract in the Reeler Mouse.

Corticospinal tract (CST) neurons are dislocated in the motor cortex of Reelin-deficient mouse, reeler. In the present study, we examined whether postnatal axonal growth arising from these dislocated CST neurons are normal or not with use of anterograde tracer, DiI and retrograde tracer, HRP. A single injection of DiI into the motor cortex of the normal and reeler mice was made during postnatal period and 8-24 hours later, the animals were sacrificed to examine DiI-labeled CST axons at the lower medulla and spinal cord. Both in the normal and reeler mice, CST axons arrived at the pyramidal decussation and entered into the contralateral spinal cord around on postnatal day (P) 0.5, and descend in the ventral area of the contralateral dorsal funiculus at C2 level on P2, at C8 level on P3, at the mid-thoracic level on P4, and at the upper lumbar level on P8. The similar results were also demonstrated by the retrograde labeling of CST neurons with injection of HRP into the C1 level or upper lumbar enlargement. Next, we examined CaMKIIα expression in the CST axons of the adult normal and reeler mice. CaMKIIα-immunopositive fibers were recognized throughout the CST pathway from the internal capsule to the dorsal funiculus of the spinal cord both in the normal and reeler mice. The present study has demonstrated that ectopic location of cell bodies of reeler CST neurons do not affect postnatal development of CST axons in the spinal cord.

Expression of Beta Subunit 2 of Ca²+/Calmodulin-Dependent Protein Kinase I in the Developing Rat Retina.

Expression of beta 2 subunit of Ca²+/calmodulin-dependent protein kinase I (CaMKIß2) of the rat retina during the developmental period and in the adulthood was studied immunohistochemically. The immunoreactivity of CaMKIß2 was detected in the earliest development of the primordial retina at embryological day (E) 12. The inner neuroblastic layer from which the presumptive ganglion cells are generated showed the ubiquitous CaMKIß2 immunoreactivity at E15 and persistently expressed at the same level until postnatal day (P) 0 when the inner neuroblastic layer divides into the ganglionic cell layer and the inner plexiform layer. The strong immunoreactivity was detected in the ganglion cell layer and the moderate one in the internal plexiform layer. CaMKIß2 immunoreactivities were persistantly expressed throughout the postnatal development at the same level. The low level of intensity was first found in the inner nuclear layer at P7, followed by the outer plexiform, outer nuclear and rod-cone cell layers at the age of P12, respectively. The intensities of CaMKIß2 immunoreactivities in the inner nuclear and rod-cone cell layers were gradually increased to the strong level by P18 and persisted until adulthood. The present study revealed that the expression of CaMKIß2 in the retina was detected from the earliest development until adulthood, indicating that CaMKIß2 may be required in both proliferation and differentiation of the retinal precursor cells and subsequent formation of the functional layers. In addition, CaMKIß2 immunoreactivity in the rod-cone cell layer implies that this protein may be involved in the visual signaling process.

Missplicing resulting from a short deletion in the reelin gene causes reeler-like neuronal disorders in the mutant shaking rat Kawasaki.

The shaking rat Kawasaki (SRK) is an autosomal recessive mutant that exhibits reeler-like abnormal locomotor behaviors. The murine reeler mutants arise from several mutations in the specific gene called reelin, which result in defects of Reelin expression or secretion in the cerebral cortex and other regions of CNS. To address the issue of whether the SRK mutation also arises from a mutation in reelin, we analyzed the reelin gene in SRK. Northern analysis of reelin mRNA from normal rats showed that rat reelin was expressed as a approximately 12-kb transcript in both the cerebrum and the cerebellum, whereas reelin expression was markedly reduced in the SRK brains. In situ hybridization analysis showed that reelin mRNA in the SRK brains was expressed in Cajal-Retzius cells in the marginal zone of the cerebral cortex and outer granular cells in the cerebellar cortex in similar manners to normal controls, but its expression was considerably reduced. On Western blotting and immunohistochemical analyses using antibodies specific for the Reelin protein, no immunoproduct was recognized in the cerebral and cerebellar cortices. From the cDNA sequences, we found a 64-base heterologous sequence in SRK reelin, which contains a termination codon in the reading frame. Furthermore, genomic DNA analysis revealed that a 10-base deletion, which contains a predicted splice donor site, occurred in the SRK genomic reelin gene, resulting in "read through" into the following intron in SRK. Thus, the SRK mutation is another type of mutation that lacks expression of the functional Reelin protein and, therefore, causes the reeler phenotype.

Reelin-expressing neurons in the anterior commissure and corpus callosum of the rat.

Reelin is an extracellular matrix protein, which plays a crucial role for the formation of laminated and nonlaminated structures in the central nervous system. To elucidate its roles in the postnatal brain, in the present study, we raised a polyclonal antibody specific for rat Reelin, and investigated Reelin-expressing neurons in the rat brain during the postnatal periods in detail. We found that some Reelin-expressing cells existed in the anterior commissure and corpus callosum. These Reelin-expressing cells were also immunostained with the antibody specific for neurons, but not immunostained with the antibodies specific for astrocytes nor oligodendrocytes, suggesting that these Reelin-expressing cells in the white matter are neurons. They are also immunostained with anti-GAD67 antibody, indicating that Reelin-expressing cells in the commissure systems are GABAergic neurons. Reelin-expressing neurons in the anterior commissure had many conspicuous varicosities on their dendritic arbors and mimic to the interfascicular neurons. These results suggest that Reelin may participate in the regulatory mechanism of neuronal activities through the commissure structure during the postnatal periods.

Subcortically and callosally projecting neurons are distinct neuronal pools in the motor cortex of the reeler mouse.

Subcortically projecting neurons and callosally projecting ones are distinct neuronal pools in the cerebral cortex of the rodents. However, cortical efferent neurons are known to project multiple targets transiently by plural collateral axons. These plural axons are eliminated during prenatal and postnatal development. In the cerebral cortex of the Reelin-deficient mouse, reeler, which is caused by mutation of the reelin gene, cortical efferent neurons are ectopically distributed. However, it is still unknown whether cortical efferent neurons in the reeler mouse lose surplus collateral axons or maintain them during developmental periods. If surplus collaterals of malpositioned cortical neurons are not eliminated, neurons projecting subcortically may project their axons to the contralateral hemisphere. To test this plausible hypothesis, we made double injections of two fluorescent dyes, Fast Blue and Diamidino yellow dihydrochloride into two of three regions, i.e., upper cervical cord, ventral lateral thalamic nucleus, and contralateral motor cortex of the normal and reeler mice, to label corticospinal, corticothalamic and callosal commissure neurons in the motor cortex, retrogradely. No double labeled neurons were identified in the motor cortex of the normal and reeler mice, although the distribution patterns of these cortical efferent neurons were completely different between normal and reeler mice. These findings strongly suggest that collateral elimination of cortical efferent neurons during developing periods are not affected in this mutant mouse.

Determination of the temperature of bremsstrahlung photon generated by ultraintense laser using various thickness attenuators.

We evaluate the simplified method using the Lambert-Beer law to measure the temperature of bremsstrahlung photon generated by an ultraintense laser. Analytical values are compared to the results of the Monte Carlo calculation of GEANT4 and they agreed very well on the condition of the appropriate distance between the attenuator and the detector. We performed the experiment to measure the temperature of bremsstrahlung x-ray emitted from a metal target irradiated by a Ti:sapphire laser with 76 mJ, 72 fs, 2.2 × 10(18) W∕cm(2). For a Cu target of 30 µm thick, the photon temperature was reasonably determined to be 0.18 MeV, which is in good agreement with previous studies.

Cortical layer V neurons in the auditory and visual cortices of normal, reeler, and yotari mice.

Both in the Reelin-deficient reeler and Dab1-deficient yotari mice, layer V corticospinal tract neurons in the sensory-motor cortex are radially spread instead of being confined to a single cortical layer. In the present study, we examined distribution pattern of cortical layer V neurons in the visual and auditory cortices of reeler and yotari mice with the injection of HRP into the superior and inferior colliculi of the adult animals, respectively. After the injection of HRP into the superior colliculus of the normal mouse, retrogradely labeled cells were distributed in layer V of the visual cortex, while the similar injection of HRP in the reeler and yotari mice produced radial dispersion of retrograde labeling through all of the depths of the visual cortex of these mutant mice. Next, we injected HRP into the inferior colliculus of the normal, reeler and yotari mice. Retrogradely labeled neurons were distributed in layer V of the normal auditory cortex, whereas they were again radially scattered in the auditory cortex of the reeler and yotari mice. Taken together with the previous and present findings, layer V cortical efferent neurons are radially scattered in the sensory-motor, visual and auditory cortices of the reeler and yotari mice.

Postnatal development of entorhinodentate projection of the Reeler mutant mouse.

We anterogradely labeled entorhinodentate axons by the injection of biotin dextran amine into the entorhinal cortex of adult wildtype and reeler mice to clarify whether the course and terminal endings of the reeler entorhinal projection are normal or not. We found that in the reeler mouse, biotin dextran amine-labeled entorhinodentate fibers arising from the entorhinal cortex curved around the hippocampal fissure instead of crossing it, whereas in the wildtype mouse, they crossed the fissure as a perforant pathway. Next, we examined carbocyanine dye (DiI) labeling of the immature entorhinodentate projection and the developmental changes of the hippocampal fissure during early postnatal days based on the laminin and glial fibrillary acidic protein (GFAP) immunohistochemistry. Injection of DiI into the entorhinal area of the wildtype and reeler mice at postnatal day 1 resulted in anterograde labeling of pioneer axons passing through the hippocampal fissure. However, follower axons could not penetrate through the hippocampal fissure in reeler mice, whereas in the normal controls, many DiI-labeled axons continued to pass through the fissure. GFAP immunohistochemistry demonstrated that GFAP-immunopositive astrocytes were abundant along the hippocampal fissure both in the wildtype and reeler mice at birth. In the wildtype mouse, GFAP-positive neurons nearby the fissure were decreasing in number during the early postnatal days, whereas in the reeler mouse, many GFAP-positive astrocytes were continuing to accumulate there. This barrier made of astrocytes in the reeler mouse may obstruct the ingrowth of the follower axons arising from the entorhinal cortex through the hippocampal fissure, resulting in the abnormal course of the entorhinodentate axons in this mutant.

Expression of zebrafish ROR alpha gene in cerebellar-like structures.

Mouse genetic studies have identified several genes involved in cerebellar development. The mouse mutants staggerer and lurcher are functionally deficient for the retinoid-related orphan receptor alpha (ROR alpha) and glutamate receptor delta2 (Grid2) genes, respectively, and they show similar functional and developmental abnormalities in the cerebellum. Here, we report the cloning and expression pattern of zebrafish ROR alpha orthologues rora1 and rora2, and compare their expression pattern with that of grid2. Expression of rora1 and rora2 is initiated at late gastrula and pharyngula stages, respectively. Both rora1 and rora2 are spatially expressed in the retina and tectum. Expression of rora2 was further observed in the cerebellum, as reported for mammalian ROR alpha. In the adult brain, rora2 and grid2 are coexpressed in brain regions, designated as cerebellar-like structures. These observations suggest an evolutionarily conserved function of ROR alpha orthologues in the vertebrate brain.