Rab6-Mediated Polarized Transport of Synaptic Vesicle Precursors Is Essential for the Establishment of Neuronal Polarity and Brain Formation.

Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6-mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

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 that EphA7 and EfnA5 are 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, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-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 STATEMENT Our 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.

Interstitial Axon Collaterals of Callosal Neurons Form Association Projections from the Primary Somatosensory to Motor Cortex in Mice.

Association projections from cortical pyramidal neurons connect disparate intrahemispheric cortical areas, which are implicated in higher cortical functions. The underlying developmental processes of these association projections, especially the initial phase before reaching the target areas, remain unknown. To visualize developing axons of individual neurons with association projections in the mouse neocortex, we devised a sparse labeling method that combined in utero electroporation and confocal imaging of flattened and optically cleared cortices. Using the promoter of an established callosal neuron marker gene that was expressed in over 80% of L2/3 neurons in the primary somatosensory cortex (S1) that project to the primary motor cortex (M1), we found that an association projection of a single neuron was the longest among the interstitial collaterals that branched out in L5 from the earlier-extended callosal projection. Collaterals to M1 elongated primarily within the cortical gray matter with little branching before reaching the target. Our results suggest that dual-projection neurons in S1 make a significant fraction of the association projections to M1, supporting the directed guidance mechanism in long-range corticocortical circuit formation over random projections followed by specific pruning.

Transient expansion of the expression region of Hsd11b1, encoding 11ß-hydroxysteroid dehydrogenase type 1, in the developing mouse neocortex.

Corticosteroids are stress-related hormones that maintain homeostasis. The most effective corticosteroids are corticosterone (CORT) in rodents and cortisol in primates. 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1; EC 1.1.1.146), encoded by Hsd11b1, is a key regulator of the local concentration of CORT/cortisol. Hsd11b1 expression in layer 5 of the primary somatosensory cortex has been shown in adult mice. However, its localization in the entire neocortex, especially during development, has not been fully addressed. Here, we established robust and dynamic expression profiles of Hsd11b1 in the developing mouse neocortex. Hsd11b1 was found mostly in pyramidal neurons. By retrograde tracing, we observed that some Hsd11b1-positive cells were projection neurons, indicating that at least some were excitatory. At postnatal day 0 (P0), Hsd11b1 was expressed in the deep layer of the somatosensory cortex. Then, from P3 to P8, the expression area expanded broadly; it was observed in layers 4 and 5, spanning the whole neocortex, including the primary motor cortex (M1) and the primary visual cortex (V1). The positive region gradually narrowed from P14 onwards and was ultimately limited to layer 5 of the somatosensory cortex at P26 and later. Furthermore, we administered CORT to nursing dams to increase the systemic CORT level of their pups. Here, we observed a reduced number of Hsd11b1-positive cells in the neocortex of these pups. Our observation suggests that Hsd11b1 expression in the developing neocortex is affected by systemic CORT levels. It is possible that stress on mothers influences the neocortical development of their children.

Loss of Rab6a in the small intestine causes lipid accumulation and epithelial cell death from lactation.

Intestinal epithelial cells (IECs) are not only responsible for the digestion and absorption of dietary substrates but also function as a first line of host defense against commensal and pathogenic luminal bacteria. Disruption of the epithelial layer causes malnutrition and enteritis. Rab6 is a small GTPase localized to the Golgi, where it regulates anterograde and retrograde transport by interacting with various effector proteins. Here, we generated mice with IEC-specific deletion of Rab6a (Rab6a∆IEC mice). While Rab6aΔIEC mice were born at the Mendelian ratio, they started to show IEC death, inflammation, and bleeding in the small intestine shortly after birth, and these changes culminated in early postnatal death. We further found massive lipid accumulation in the IECs of Rab6a∆IEC neonates. In contrast to Rab6a∆IEC neonates, knockout embryos did not show any of these abnormalities. Lipid accumulation and IEC death became evident when Rab6a∆IEC embryos were nursed by a foster mother, suggesting that dietary milk-derived lipids accumulated in Rab6a-deficient IECs and triggered IEC death. These results indicate that Rab6a plays a crucial role in regulating the lipid transport and maintaining tissue integrity.

Epiphycan is specifically expressed in cochlear supporting cells and is necessary for normal hearing.

The study of inner ear specific transcripts has revealed novel information about hereditary hearing loss and a mechanism of normal hearing. In this study, by analyzing a published cDNA library, we focused on Epiphycan (Epyc), a member of the small leucine-rich repeat proteoglycan family, whose transcript is enriched in the inner ear. Epyc mRNA was expressed abundantly and specifically in adult mice cochleae and was localized in supporting cells within the organ of Corti of both neonatal and adult mice. To examine the function of Epyc, we generated Epyc knockout (KO) mice using the CRISPR/Cas9 system. Epyc KO mice cochleae exhibited normal morphology. However, measurement of the auditory brain-stem response in Epyc KO mice revealed an elevated hearing threshold above 16 kHz frequency. This study suggests that Epyc is necessary for normal auditory function.

Echocardiographic Estimates of Left Ventricular Diastolic Dysfunction Do Not Predict the Clinical Course in Elderly Patients Undergoing Transcatheter Atrial Septal Defect Closure: Impact of Early Diastolic Mitral Annular Velocity.

OBJECTIVE: This study evaluated the effect of echocardiographic left ventricular (LV) diastolic dysfunction on acute congestive heart failure after transcatheter atrial septal defect (ASD) closure in elderly patients. BACKGROUND: Although there is concern that LV diastolic dysfunction develops acute congestive heart failure after ASD closure, limited information is available regarding the influence, especially in elderly patients with severe LV diastolic dysfunction. METHODS: Two hundred consecutive patients older than 60 years were divided into 3 groups according to echocardiographic LV diastolic dysfunction: severe (early diastolic mitral annular velocity [e'] <5.0 cm/s), mild (5.0≤ e' <8.0 cm/s), and normal (e' ≥ 8.0 cm/s). Changes in plasma B-type natriuretic peptide (BNP) levels were evaluated. RESULTS: No patients with severe LV diastolic dysfunction developed acute congestive heart failure immediately after the procedure. BNP levels unchanged after the procedure in patients with severe LV diastolic dysfunction (126 ± 181 to 131 ± 148 pg/ml, P = 0.885), and this increase in BNP levels was not different from that between the diagnosis of ASD and the procedure. The change in BNP levels in patients with severe LV diastolic dysfunction, who were frequently treated with diuretics before the procedure, was equivalent to that in patients with mild LV diastolic dysfunction and normal LV diastolic function (5 ± 119 vs. 16 ± 101 vs. 9 ± 131 pg/ml, P = 0.724). CONCLUSIONS: Our findings suggest that transcatheter ASD closure under volume management is safe and valuable in elderly patients with echocardiographic severe LV diastolic dysfunction.

DBZ regulates cortical cell positioning and neurite development by sustaining the anterograde transport of Lis1 and DISC1 through control of Ndel1 dual-phosphorylation.

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 GSK3ß 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 GSK3ß 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.

Sgk1 regulates desmoglein 1 expression levels in oligodendrocytes in the mouse corpus callosum after chronic stress exposure.

Major depression, one of the most prevalent mental illnesses, is thought to be a multifactorial disease related to both genetic and environmental factors. However, the genes responsible for and the pathogenesis of major depression at the molecular level remain unclear. Recently, we reported that stressed mice with elevated plasma corticosterone levels show upregulation and activation of serum glucocorticoid-regulated kinase (Sgk1) in oligodendrocytes. Active Sgk1 causes phosphorylation of N-myc downstream-regulated gene 1 (Ndrg1), and phospho-Ndrg1 increases the expression of N-cadherin, α-catenin, and ß-catenin in oligodendrocytes. This activation of the Sgk1 cascade results in morphological changes in the oligodendrocytes of nerve fiber bundles, such as those present in the corpus callosum. However, little is known about the molecular functions of the traditional and/or desmosomal cadherin superfamily in oligodendrocytes. Therefore, in this study, we aimed to elucidate the functions of the desmosomal cadherin superfamily in oligodendrocytes. Desmoglein (Dsg) 1, Dsg2, and desmocollin 1 (Dsc1) were found to be expressed in the corpus callosum of mouse brain, and the expression of a subtype of Dsg1, Dsg1c, was upregulated in oligodendrocytes after chronic stress exposure. Furthermore, Dsg1 proteins were localized around the plasma membrane regions of oligodendrocytes. A study in primary oligodendrocyte cultures also revealed that chronic upregulation of Sgk1 by dexamethasone administration is involved in upregulation of Dsg1c mRNA. These results may indicate that chronic stress induced Sgk1 activation in oligodendrocytes, which increases Dsg1 expression near the plasma membrane. Thus, Dsg1 upregulation may be implicated in the molecular mechanisms underlying the morphological changes in oligodendrocytes in response to chronic stress exposure.

Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.

Long-term effects of transcatheter closure of atrial septal defect on cardiac remodeling and exercise capacity in patients older than 40 years with a reduction in cardiopulmonary function.

BACKGROUND: Although it has been demonstrated that cardiac remodeling and exercise capacity improve after transcatheter closure of atrial septal defect (ASD), little is known about long-term benefits in middle-aged and elderly patients with a reduction in cardiopulmonary function. OBJECTIVES: To evaluate long-term extent and time course of improvements in cardiac remodeling and exercise capacity in those patients. METHODS: Twenty ASD patients ≥ 40 years of age with a reduction in cardiopulmonary function (predicted peak oxygen uptake [VO(2)] < 65%) were enrolled. Transthoracic echocardiography and cardiopulmonary exercise testing were performed at baseline and at 1 month, 3 months, 6 months, and >12 months after the procedure. RESULTS: At 1 month after the procedure, significant decreases in right ventricular (RV) end-diastolic diameter (38.2 ± 4.4 to 31.9 ± 4.4 mm; P < 0.001) and RV/left ventricular end-diastolic diameter ratio (0.95 ± 0.17 to 0.71 ± 0.13; P < 0.001) occurred, and they were maintained during the follow-up period. Normal RV size was achieved in 11 of 18 patients with RV enlargement. Predicted peak VO(2) did not change at 1 month and 3 months, but it improved significantly after 6 months (53.6 ± 6.5 to 62.1 ± 12.6%; P < 0.01). Sixteen of the 20 patients showed improved predicted peak VO(2). CONCLUSIONS: Cardiac remodeling and exercise capacity could be improved over the long-term period after transcatheter closure of ASD in middle-aged and elderly patients with a reduction in cardiopulmonary function. There were differences in the time course of improvement between cardiac remodeling and exercise capacity in those patients.

Direct contact of fibroblasts with neuronal processes promotes differentiation to myofibroblasts and induces contraction of collagen matrix in vitro.

Wound healing is often delayed in the patients whose sensory and autonomic innervation is impaired. We hypothesized that existence of neurites in the skin may promote wound healing by inducing differentiation of fibroblasts into myofibroblasts with consequent wound contraction. In the current study, we examined the effect of neurons on differentiation of fibroblasts and contraction of collagen matrix in vitro using a new co-culture model. Neuronal cell line, PC12 cells, of which the neurite outgrowth can be controlled by adding nerve growth factor, was used. Rat dermal fibroblasts were co-cultured with PC12 cells extending neurites or with PC12 cells lacking neurites. Then, differentiation of fibroblasts into myofibroblasts and contraction of the collagen matrix was evaluated. Finally, we examined whether direct or indirect contact with neurites of PC12 cells promoted the differentiation of fibroblasts. Our results showed that fibroblasts co-cultured with PC12 extending neurites differentiated into myofibroblasts more effectively and contracted the collagen matrix stronger than those with PC12 lacking neurites. Direct contact of fibroblasts with neurites promoted more differentiation than indirect contact. In conclusion, direct contact of fibroblasts with neuronal processes is important for differentiation into myofibroblasts and induction of collagen gel contraction, leading to promotion of wound healing.

Evaluation of exercise capacity using wave intensity in chronic heart failure with normal ejection fraction.

Impaired exercise capacity has been found in patients with diastolic dysfunction with preserved systolic function. Although conventional transthoracic echocardiography (TTE) provides useful clinical information about systolic and diastolic cardiac function, its capability to evaluate exercise capacity has been controversial. The inertia force of late systolic aortic flow is known to have a tight relationship with left ventricular (LV) performance during the period from near end-systole to isovolumic relaxation. The inertia force and the time constant of LV pressure decay during isovolumic relaxation can be estimated noninvasively using the second peak (W(2)) of wave intensity (WI), which is measured with an echo-Doppler system. We sought to determine whether W(2) is associated with exercise capacity in patients with chronic heart failure with normal ejection fraction (HFNEF) and to compare its ability to predict exercise capacity with parameters obtained by conventional TTE including tissue Doppler imaging. Sixteen consecutive patients with chronic HFNEF were enrolled in this study. Wave intensity was obtained with a color Doppler system for measurement of blood velocity combined with an echo-tracking system for detecting changes in vessel diameter. Concerning conventional TTE, we measured LV ejection fraction (EF), peak velocities of early (E) and late (A) mitral inflow using pulse-wave Doppler, and early (Ea) and late (Aa) diastolic velocities using tissue Doppler imaging. Left ventricular EF, E/A ratio, Ea, and E/Ea ratio did not correlate with exercise capacity, whereas W(2) significantly correlated with peak VO(2) (r = 0.54, p = 0.03), VE/VCO(2) slope (r = -0.53, p = 0.03), and ΔVO(2)/ΔWR (r = 0.56, p = 0.02). W(2) was associated with exercise capacity in patients with chronic HFNEF. In conclusion, W(2) is considered to be clinically more useful than conventional TTE indices for evaluating exercise capacity in patients with chronic HFNEF.

EHBP1L1, an apicobasal polarity regulator, is critical for nuclear polarization during enucleation of erythroblasts.

Cell polarity, the asymmetric distribution of proteins and organelles, is permanently or transiently established in various cell types and plays an important role in many physiological events. epidermal growth factor receptor substrate 15 homology domain-binding protein 1-like 1 (EHBP1L1) is an adapter protein that is localized on recycling endosomes and regulates apical-directed transport in polarized epithelial cells. However, the role of EHBP1L1 in nonepithelial cells, remains unknown. Here, Ehbp1l1-/- mice showed impaired erythroblast enucleation. Further analyses showed that nuclear polarization before enucleation was impaired in Ehbp1l1-/- erythroblasts. It was also revealed that EHBP1L1 interactors Rab10, Bin1, and dynamin were involved in erythroblast enucleation. In addition, Ehbp1l1-/- erythrocytes exhibited stomatocytic morphology and dehydration. These defects in erythroid cells culminated in early postnatal anemic lethality in Ehbp1l1-/- mice. Moreover, we found the mislocalization of nuclei and mitochondria in the skeletal muscle cells of Ehbp1l1-/- mice, as observed in patients with centronuclear myopathy with genetic mutations in Bin1 or dynamin 2. Taken together, our findings indicate that the Rab8/10-EHBP1L1-Bin1-dynamin axis plays an important role in multiple cell polarity systems in epithelial and nonepithelial cells.

Transcatheter closure of atrial septal defect in a geriatric population.

OBJECTIVES: To evaluate the efficacy and safety of transcatheter closure of atrial septal defects (ASD) in patients over 70 years of age. BACKGROUND: Transcatheter closure of ASD is an established procedure in children and young adults, but the benefits of this procedure in geriatric patients are still unclear. METHODS: Between 2005 and 2010, 430 patients with ASD underwent transcatheter closure in our hospital. Among those patients, 30 consecutive patients older than 70 years of age were prospectively evaluated. RESULTS: Mean age at procedure was 75.8 ± 3.8 years (range: 70-85 years). Mean Qp/Qs was 2.4 ± 0.7 and mean ASD diameter was 20.3 ± 6.4 mm. Nine patients (30%) had a history of hospitalization due to heart failure. ASD closure was successfully performed in 28 patients (93%) without significant complications. During the follow-up period (mean period of 19.1 ± 11.3 months), New York Heart Association (NYHA) functional class was significantly improved in 20 patients (74%). Significant improvements of plasma BNP level, resting heart rate, and systolic pulmonary artery pressure were also observed. Improvement of tricuspid regurgitation was observed in 11 of 17 patients with moderate or severe regurgitation during the follow-up period. Conversely, worsening of mitral regurgitation was observed in 10 of the 27 patients. CONCLUSION: Transcatheter closure of ASD in geriatric patients can be performed safely. This procedure contributes to significant improvement of symptoms and positive cardiac remodeling. Long-term follow-up is mandatory, especially for patients with mitral regurgitation.

Transcatheter closure of a large atrial septal defect under microprobe transesophageal echocardiographic guidance.

We present a case of an atrial septal defect (ASD) in a 59-year-old man with an indication for ASD closure who also had a history of chronic obstructive pulmonary disease. Because of his decreased respiratory function with multiple bullae in his lungs, the procedure was performed without general anesthesia under the guidance of fluoroscopy and two-dimensional (2D) transesophageal echocardiography (TEE) using a transesophageal echocardiographic microprobe (micro-TEE) (S8-3t; Philips Medical Systems, Andover, MA, USA). The micro-TEE probe was inserted into the esophagus smoothly and easily in the supine position without sedation. It revealed a deficient superior-anterior rim and adequate rims elsewhere, and the maximal diameter of ASD was measured to be 25 mm. Balloon sizing resulted in a stretched defect diameter of 29 mm using the stop-flow technique. A 30-mm AMPLATZER Septal Occluder (AGA Medical, Plymouth, MN, USA) was deployed. The micro-TEE demonstrated that both disks were on the appropriate sides of the interatrial septum and the device was not interfering with surround cardiac structures. Residual shunt flow was not detected with color Doppler. The device was released successfully without any complications. Recently introduced multiplane micro-TEE can provide adequate information about a large ASD with a less invasive procedure in adult patients. Micro-TEE has a potential to become a novel imaging option for interventions of the interatrial septum.

Catheter closure of coronary sinus atrial septal defect using Amplatzer Septal Occluder.

Coronary sinus defect is a rare type of atrial septal defect. We report two patients who had a coronary sinus atrial septal defect without persistent left superior caval vein, where the orifice of the coronary sinus was closed using the Amplatzer Septal Occluder. The procedure was successful, without any complications including conduction disturbance.

Cytoneme-like protrusion formation induced by LAR is promoted by receptor dimerization.

Actin-based protrusions called cytonemes are reported to function in cell communication by supporting events such as morphogen gradient establishment and pattern formation. Despite the crucial roles of cytonemes in cell signaling, the molecular mechanism for cytoneme establishment remains elusive. In this study, we showed that the leukocyte common antigen-related (LAR) receptor protein tyrosine phosphatase plays an important role in cytoneme-like protrusion formation. Overexpression of LAR in HEK293T cells induced the formation of actin-based protrusions, some of which exceeded 200 µm in length and displayed a complex morphology with branches. Upon focusing on the regulation of LAR dimerization or clustering and the resulting regulatory effects on LAR phosphatase activity, we found that longer and more branched protrusions were formed when LAR dimerization was artificially induced and when heparan sulfate was applied. Interestingly, although the truncated form of LAR lacking phosphatase-related domains promoted protrusion formation, the phosphatase-inactive forms did not show clear changes, suggesting that LAR dimerization triggers the formation of cytoneme-like protrusions in a phosphatase-independent manner. Our results thus emphasize the importance of LAR and its dimerization in cell signaling. This article has an associated First Person interview with the first author of the paper.

Fezf2-positive fork cell-like neurons in the mouse insular cortex.

The fork cell and von Economo neuron, which are found in the insular cortex and/or the anterior cingulate cortex, are defined by their unique morphologies. Their shapes are not pyramidal; the fork cell has two primary apical dendrites and the von Economo neurons are spindle-shaped (bipolar). Presence of such neurons are reported only in the higher animals, especially in human and great ape, indicating that they are specific for most evolved species. Although it is likely that these neurons are involved in higher brain function, lack of results with experimental animals makes further investigation difficult. We here ask whether equivalent neurons exist in the mouse insular cortex. In human, Fezf2 has been reported to be highly expressed in these morphologically distinctive neurons and thus, we examined the detailed morphology of Fezf2-positive neurons in the mouse brain. Although von Economo-like neurons were not identified, Fezf2-positive fork cell-like neurons with two characteristic apical dendrites, were discovered. Examination with electron microscope indicated that these neurons did not embrace capillaries, rather they held another cell. We here term such neurons as holding neurons. We further observed several molecules, including neuromedin B (NMB) and gastrin releasing peptide (GRP) that are known to be localized in the fork cells and/or von Economo cells in human, were localized in the mouse insular cortex. Based on these observations, it is likely that an equivalent of the fork cell is present in the mouse.

Pericentrin, a centrosomal protein related to microcephalic primordial dwarfism, is required for olfactory cilia assembly in mice.

The Drosophila pericentrin-like protein has been shown to be essential for the formation of the sensory cilia of chemosensory and mechanosensory neurons by mutant analysis in flies, while the in vivo function of pericentrin, a well-studied mammalian centrosomal protein related to microcephalic primordial dwarfism, has been unclear. To determine whether pericentrin is required for ciliogenesis in mammals, we generated and analyzed mice with a hypomorphic mutation of Pcnt encoding the mouse pericentrin. Immunofluorescence analysis demonstrated that olfactory cilia of chemosensory neurons in the nasal olfactory epithelium were malformed in the homozygous mutant mice. On the other hand, the assembly of motile and primary cilia of non-neuronal epithelial cells and the formation of sperm flagella were not affected in the Pcnt-mutant mice. The defective assembly of olfactory cilia in the mutant was apparent from birth. The mutant animals displayed reduced olfactory performance in agreement with the compromised assembly of olfactory cilia. Our findings suggest that pericentrin is essential for the assembly of chemosensory cilia of olfactory receptor neurons, but it is not globally required for cilia formation in mammals.