Image recognition-based petal arrangement estimation.

Flowers exhibit morphological diversity in the number and positional arrangement of their floral organs, such as petals. The petal arrangements of blooming flowers are represented by the overlap position relation between neighboring petals, an indicator of the floral developmental process; however, only specialists are capable of the petal arrangement identification. Therefore, we propose a method to support the estimation of the arrangement of the perianth organs, including petals and tepals, using image recognition techniques. The problem for realizing the method is that it is not possible to prepare a large number of image datasets: we cannot apply the latest machine learning based image processing methods, which require a large number of images. Therefore, we describe the tepal arrangement as a sequence of interior-exterior patterns of tepal overlap in the image, and estimate the tepal arrangement by matching the pattern with the known patterns. We also use methods that require less or no training data to implement the method: the fine-tuned YOLO v5 model for flower detection, GrubCut for flower segmentation, the Harris corner detector for tepal overlap detection, MAML-based interior-exterior estimation, and circular permutation matching for tepal arrangement estimation. Experimental results showed good accuracy when flower detection, segmentation, overlap location estimation, interior-exterior estimation, and circle permutation matching-based tepal arrangement estimation were evaluated independently. However, the accuracy decreased when they were integrated. Therefore, we developed a user interface for manual correction of the position of overlap estimation and interior-exterior pattern estimation, which ensures the quality of tepal arrangement estimation.

Identification of potent siRNA targeting complement C5 and its robust activity in pre-clinical models of myasthenia gravis and collagen-induced arthritis.

Complement component 5 (C5), an important molecule in the complement cascade, blockade by antibodies shows clinical efficacy in treating complement-mediated disorders. However, insufficient blockading induced by single-nucleotide polymorphisms in the C5 protein or frequent development of "breakthrough" intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria treated with eculizumab have been reported. Herein, we developed a lipid nanoparticle (LNP)-formulated siRNA targeting C5 that was efficiently delivered to the liver and silenced C5 expression. We identified a potent C5-siRNA with an in vitro IC50 of 420 pM and in vivo ED50 of 0.017 mg/kg following a single administration. Single or repeated administrations of the LNP-formulated C5-siRNA allowed robust and durable suppression of liver C5 expression in mice. Complement C5 silencing ameliorated C5b-dependent anti-acetylcholine receptor antibody-induced myasthenia gravis and C5a-dependent collagen-induced arthritis symptoms. Similarly, in nonhuman primates, a single administration of C5-siRNA/LNP-induced dose-dependent plasma C5 suppression and concomitantly inhibited serum complement activity; complement activity recovered to the pre-treatment levels at 65 days post administration, thus indicating that the complement activity can be controlled for a specific period. Our findings provide the foundation for further developing C5-siRNA delivered via LNPs as a potential therapeutic for complement-mediated diseases.

The Effect of Geometrical Overlap between Giant Magnetoresistance Sensor and Magnetic Flux Concentrators: A Novel Comb-Shaped Sensor for Improved Sensitivity.

The combination of magnetoresistive (MR) element and magnetic flux concentrators (MFCs) offers highly sensitive magnetic field sensors. To maximize the effect of MFC, the geometrical design between the MR element and MFCs is critical. In this paper, we present simulation and experimental studies on the effect of the geometrical relationship between current-in-plane giant magnetoresistive (GMR) element and MFCs made of a NiFeCuMo film. Finite element method (FEM) simulations showed that although an overlap between the MFCs and GMR element enhances their magneto-static coupling, it can lead to a loss of magnetoresistance ratio due to a magnetic shielding effect by the MFCs. Therefore, we propose a comb-shaped GMR element with alternate notches and fins. The FEM simulations showed that the fins of the comb-shaped GMR element provide a strong magneto-static coupling with the MFCs, whereas the electric current is confined within the main body of the comb-shaped GMR element, resulting in improved sensitivity. We experimentally demonstrated a higher sensitivity of the comb-shaped GMR sensor (36.5 %/mT) than that of a conventional rectangular GMR sensor (28 %/mT).

Novel Complement C5 Small-interfering RNA Lipid Nanoparticle Prolongs Graft Survival in a Hypersensitized Rat Kidney Transplant Model.

BACKGROUND: Prophylaxis of antibody-mediated rejection (AMR) caused by donor-specific antibodies remains challenging. Given the critical roles of complement activity in antibody-mediated graft injury, we developed a lipid nanoparticle (LNP) formulation of small-interfering RNA against complement C5 (C5 siRNA-LNP) and investigated whether C5 siRNA-LNP could downregulate the complement activity and act as an effective treatment for AMR. METHODS: Lewis recipient rats were sensitized by skin grafting from Brown Norway donor rats. Kidney transplantation was performed at 4 wk post-skin grafting.C5 siRNA- or control siRNA-LNP was administered intravenously, and the weekly injections were continued until the study's conclusion. Cyclosporin (CsA) and/or deoxyspergualin (DSG) were used as adjunctive immunosuppressants. Complement activity was evaluated using hemolysis assays. The deposition of C5b9 in the grafts was evaluated using immunohistochemical analysis on day 7 posttransplantation. RESULTS: C5 siRNA-LNP completely suppressed C5 expression and complement activity (hemolytic activity ≤ 20%) 7 d postadministration. C5 siRNA-LNP in combination with CsA and DSG (median survival time: 56.0 d) prolonged graft survival compared with control siRNA-LNP in combination with CsA and DSG (median survival time: 21.0 d; P = 0.0012; log-rank test). Immunohistochemical analysis of the grafts revealed that downregulation of C5 expression was associated with a reduction in C5b9-positive area ( P = 0.0141, Steel-Dwass test). CONCLUSIONS: C5 siRNA-LNP combined with immunosuppressants CsA and DSG downregulated C5 activity and significantly prolonged graft survival compared with control siRNA-LNP with CsA and DSG. Downregulation of C5 expression using C5 siRNA-LNP may be an effective therapeutic approach for AMR.

Blockade of the fractalkine-CX3CR1 axis ameliorates experimental colitis by dislodging venous crawling monocytes.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN-CX3CR1 axis's role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN-CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/- monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN-CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN-CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

Role of Anti-Fractalkine Antibody in Suppression of Joint Destruction by Inhibiting Migration of Osteoclast Precursors to the Synovium in Experimental Arthritis.

OBJECTIVE: To elucidate the role of the fractalkine (FKN)/CX3 CR1 pathway in joint destruction in rheumatoid arthritis. METHODS: We examined the effect of treatment with anti-mouse FKN (anti-mFKN) monoclonal antibody (mAb) on joint destruction and the migration of osteoclast precursors (OCPs) into the joint, using the collagen-induced arthritis (CIA) model. DBA/1 mice were immunized with bovine type II collagen to induce arthritis, and then treated with anti-mFKN mAb. Disease severity was monitored by arthritis score, and joint destruction was evaluated by soft x-ray and histologic analyses. Plasma levels of joint destruction markers were assessed by enzyme-linked immunosorbent assay. FKN expression on endothelial cells was detected by immunohistochemistry. Bone marrow-derived OCPs were labeled with fluorescein and transferred to mice with CIA, and the migration of the OCPs to the joints was then analyzed. RESULTS: Both prophylactic and therapeutic treatment with anti-mFKN mAb significantly decreased the arthritis and soft x-ray scores. Plasma levels of cartilage oligomeric matrix protein and matrix metalloproteinase 3 decreased after treatment with anti-mFKN mAb. Histologic analysis revealed that anti-mFKN mAb inhibited synovitis, pannus formation, and cartilage destruction, as well as suppressed bone damage, with a marked reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. Anti-mFKN mAb strongly inhibited the migration of bone marrow-derived OCPs into the affected synovium. CONCLUSION: Anti-mFKN mAb notably ameliorates arthritis and joint destruction in the CIA model, as well as inhibits migration of OCPs into the synovium. These results suggest that inhibition of the FKN/CX3 CR1 pathway could be a novel strategy for treatment of both synovitis and joint destruction in rheumatoid arthritis.

Cataract-preventing contact lens for in vivo imaging of mouse retina.

In vivo imaging of mouse retinas using two-photon or confocal laser scanning fluorescence microscopy is a powerful tool for time-lapse analyses of the dynamic movements of cell populations. However, acute and reversible opacification of the crystalline lenses of mouse eyes under anesthesia decreases the visibility of the ocular fundus. Therefore, we developed a customized contact lens for preventing cataract during continuous retinal imaging in anesthetized mice. This experimental approach will aid in the elucidation of cellular and molecular dynamics in the CNS under physiological and pathological conditions.

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine.

Temporal identity transition from Purkinje cell progenitors to GABAergic interneuron progenitors in the cerebellum.

In the cerebellum, all GABAergic neurons are generated from the Ptf1a-expressing ventricular zone (Ptf1a domain). However, the machinery to produce different types of GABAergic neurons remains elusive. Here we show temporal regulation of distinct GABAergic neuron progenitors in the cerebellum. Within the Ptf1a domain at early stages, we find two subpopulations; dorsally and ventrally located progenitors that express Olig2 and Gsx1, respectively. Lineage tracing reveals the former are exclusively Purkinje cell progenitors (PCPs) and the latter Pax2-positive interneuron progenitors (PIPs). As development proceeds, PCPs gradually become PIPs starting from ventral to dorsal. In gain- and loss-of-function mutants for Gsx1 and Olig1/2, we observe abnormal transitioning from PCPs to PIPs at inappropriate developmental stages. Our findings suggest that the temporal identity transition of cerebellar GABAergic neuron progenitors from PCPs to PIPs is negatively regulated by Olig2 and positively by Gsx1, and contributes to understanding temporal control of neuronal progenitor identities.

The c-Ski family member and transcriptional regulator Corl2/Skor2 promotes early differentiation of cerebellar Purkinje cells.

Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture, and loss of PCs causes severe cerebellar dysfunction. The mechanisms underlying cell fate determination and early differentiation of PC remain largely unknown. Here we show that the c-Ski family member and transcriptional regulator Corl2 is required for correct differentiation of PCs. In Corl2 knock-out embryos, initial PC specification appeared largely normal, but in a subset of presumptive PCs generated near the ventral border of the PC domain, cell fate choice was compromised and cells showed a mixed identity expressing the interneuron marker Pax2 as well. Additionally, selection and maintenance of the transmitter phenotype was compromised in most developing PCs in the mutants. During later differentiation steps, induction of PC marker genes was significantly suppressed, suggesting that maturation was delayed in the absence of Corl2. Consistently, defects in migration, cell polarization and dendrite formation were observed in mutant PCs, although their axonal trajectories appeared normal. These phenotypes closely resembled those of mutants for Rora, an essential regulator of PC differentiation. However, Rora expression was not significantly changed in the Corl2 mutants, indicating that Corl2 does not simply act upstream of Rora to promote PC differentiation. ChIP experiments revealed that Corl2 bound to the promoter regions of several PC-selective genes, which are also known to be direct downstream targets of RORα. Altogether, our results identified a novel regulatory program of PC differentiation involving Corl2, which might cooperate with the RORα pathway.

Necl-2/CADM1 interacts with ErbB4 and regulates its activity in GABAergic neurons.

The neuronal network is tightly regulated by a large variety of locally connected GABAergic neurons. Neuregulin1 (Nrg1) and its receptor ErbB4 are master regulators in the morphological and functional development of excitatory synapses in GABAergic neurons. We previously showed that the immunoglobulin-like cell adhesion molecule, nectin-like molecule-2 (Necl-2)/CADM1, interacts with the ErbB3 and ErbB4 receptors, and that the interaction of Necl-2 with ErbB3 inhibits the Nrg1-induced ErbB3/ErbB2 signaling in epithelial cells. Here, we examined the role of the interaction of Necl-2 with ErbB4 in GABAergic neurons. Necl-2 was co-expressed with ErbB4 in parvalbumin-positive GABAergic neurons in the mouse hippocampus and co-localized with ErbB4 at excitatory synapses. Necl-2 knockdown enhanced the Nrg1-induced phosphorylation of ErbB4. Moreover, overexpression of PTPN13, which is a tyrosine phosphatase bound to the cytoplasmic tail of Necl-2, suppressed the Nrg1-induced development of excitatory synapses in GABAergic neurons through the inhibition of ErbB4 activity. These results indicate that Necl-2 interacts with ErbB4 and regulates the development of excitatory synapses via the regulation of ErbB4 activity in GABAergic neurons.

Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5alpha/beta.

LL5beta has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)-bound microtubule plus ends to the cell cortex. In this study, we show that LL5beta and its homologue LL5alpha (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins alpha3beta1 and alpha6beta4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin alpha3 at the basal cell cortex. Activation of integrin alpha3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin-integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.

The basic helix-loop-helix transcription factor Nato3 controls neurogenic activity in mesencephalic floor plate cells.

Floor plate (FP) cells, the ventral midline cells of the developing neural tube, have long been thought to be non-neurogenic organizer cells that control neuronal patterning and axonal guidance. Recent studies have revealed that mesencephalic FP (mesFP) cells have neurogenic activity and generate dopaminergic neurons. However, the mechanisms underlying the control of neurogenic potential in FP cells are not yet fully understood. Here we identified the bHLH factor Nato3 as an FP-specific transcription factor. In Nato3-null mutant mice, FP cells in the spinal cord were correctly specified, but could not properly mature. By contrast, in the developing mesencephalon, loss of Nato3 did not affect FP differentiation, but led to loss of neurogenic activity in the medial subpopulation of mesFP cells by suppressing proneural gene expression and inducing cell cycle arrest. As a consequence, the number of midbrain dopaminergic neurons generated was decreased in mutants. We also found that Hes1, which is known to be required for non-dividing organizer cell development in the neural tube, was aberrantly upregulated in the mesFP cells of Nato3 mutants. Consistently, forced expression of Nato3 repressed Hes1 expression and consequently induced premature neurogenesis. Finally, we showed that forced expression of Hes1 in mesFP cells induced cell cycle arrest and downregulation of proneural factors. Taken together, these results suggest that Nato3 confers neurogenic potential on mesFP cells by suppressing classical non-neurogenic FP cell differentiation, at least in part, through repressing Hes1.

Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin.

GABAergic Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture. Although the mechanisms of PC differentiation have been well studied, the precise origin and initial specification mechanism of PCs remain to be clarified. Here, we identified a cerebellar and spinal cord GABAergic progenitor-selective cell surface marker, Neph3, which is a direct downstream target gene of Ptf1a, an essential regulator of GABAergic neuron development. Using FACS, Neph3(+) GABAergic progenitors were sorted from the embryonic cerebellum, and the cell fate of this population was mapped by culturing in vitro. We found that most of the Neph3(+) populations sorted from the mouse E12.5 cerebellum were fated to differentiate into PCs while the remaining small fraction of Neph3(+) cells were progenitors for Pax2(+) interneurons, which are likely to be deep cerebellar nuclei GABAergic neurons. These results were confirmed by short-term in vivo lineage-tracing experiments using transgenic mice expressing Neph3 promoter-driven GFP. In addition, we identified E-cadherin as a marker selectively expressed by a dorsally localized subset of cerebellar Neph3(+) cells. Sorting experiments revealed that the Neph3(+) E-cadherin(high) population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2(+) interneurons were enriched in the Neph3(+) E-cadherin(low) population. Taken together, our results identify two spatially demarcated subregions that generate distinct cerebellar GABAergic subtypes and reveal the origin of PCs in the ventricular zone of the cerebellar primordium.

Lmx1a and Lmx1b cooperate with Foxa2 to coordinate the specification of dopaminergic neurons and control of floor plate cell differentiation in the developing mesencephalon.

Mesencephalic dopaminergic (mesDA) neurons control movement and behavior, and their loss causes severe neurological disorders, such as Parkinson's disease. Recent studies have revealed that mesDA neurons originate from mesencephalic floor plate (FP) cells, which had been thought of as non-neurogenic organizer cells regulating regional patterning and axonal projections. Otx2 and its FP-specific downstream factor Lmx1a have been shown to be sufficient to confer neurogenic activity on FP cells and determine a mesDA fate. However, the mechanism underlying how these factors control mesDA development and how FP cells and mesDA neurons are coordinately specified are still largely unknown. In the present study, we obtained evidence that Lmx1a and Lmx1b cooperate with Foxa2 to specify mesDA neuron identity by gain-of-function approaches using transgenic mice. Lmx1a/b appeared to select a mesDA fate by suppressing red nucleus fate in the context of Foxa2-positive progenitors, at least in part, through repressing the Sim1-Lhx1 and Ngn1 pathways that inhibit proper mesDA differentiation. We also found that, in the mesencephalon, FP cell fate is primarily determined by Foxa2 with a supportive action of Lmx1a/b through repressing Nkx6.1, which inhibits FP cell differentiation. Thus, FP and mesDA identities are determined by distinct specification pathways, both of which are controlled by the same combination of transcription factors, Lmx1a/b and Foxa2, and, as a consequence, mesDA neurons are generated from mesencephalic FP cells.

Identification of a novel transcriptional corepressor, Corl2, as a cerebellar Purkinje cell-selective marker.

The developmental origin of cerebellar Purkinje cells (PCs) has not been precisely mapped and the genetic program of the specification of this neuronal subtype is largely unknown. Here, we report the isolation of a novel mouse gene encoding a transcriptional corepressor, Corl2, and its expression pattern. Corl2 expression was restricted to the central nervous system in both adult and embryonic stages. In situ hybridization and immunohistochemistry using a polyclonal antibody against Corl2 revealed that Corl2 is selectively expressed at high levels in the developing cerebellum, ventral metencephalon and myelencephalon at E12.5. In these brain regions, neural progenitors did not express Corl2 during the proliferative state but started to express it shortly after exit from the cell cycle. In the cerebellum, Corl2 was specifically expressed in PCs at the adult stage, and consistently, most Corl2(+) cells expressed PC markers, such as RORalpha and calbindin, at the late embryonic stage. At E12.5, when PCs are emerging, GABAergic neurons generated from the dorsal part of the Ptf1a(+) progenitor domain selectively expressed Corl2. Importantly, Corl2(+) cells in the cerebellum did not express the GABAergic interneuron marker Pax2 at any of the developmental stages. Collectively, these results strongly suggest that Corl2 is a specific marker for PCs in the cerebellum from their emergence until the adult stage. Furthermore, this marker was useful for unmasking the precise origin of PCs and delineating the domain map within the ventricular zone that generates cerebellar GABAergic neurons.

Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells.

Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinson's disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinson's disease.

Helt determines GABAergic over glutamatergic neuronal fate by repressing Ngn genes in the developing mesencephalon.

The mechanism underlying the determination of neurotransmitter phenotype in the developing mesencephalon, particularly GABAergic versus glutamatergic fate, remains largely unknown. Here, we show in mice that the basic helix-loop-helix transcriptional repressor gene Helt (also known as Megane and Heslike) functions as a selector gene that determines GABAergic over glutamatergic fate in the mesencephalon. Helt was coincidently expressed in all the progenitor domains for mesencephalic GABAergic neurons. In the mesencephalon of Helt-deficient embryos, GABAergic neurons were mostly absent and glutamatergic neurons emerged instead. Conversely, ectopically expressed Helt suppressed glutamatergic formation and induced GABAergic neurogenesis. However, the Helt mutants showed normal progenitor domain formation. In consequence, postmitotic expression of the homeodomain factor Nkx2.2, which was specifically expressed by GABAergic populations in wild-type embryos, was maintained despite the transmitter phenotype conversion from GABAergic to glutamatergic in the Helt mutants, suggesting that Helt is not involved in neuronal identity specification. Furthermore, we identified proneural genes Ngn1 and Ngn2, which were selectively expressed in glutamatergic progenitors in the developing mesencephalon and had the ability to confer the glutamatergic fate, as downstream target genes of Helt. These results suggest that Helt determines GABAergic over glutamatergic fate, at least in part, by repressing Ngn (Neurog) genes and that basic helix-loop-helix transcription factor networks involving Helt and Ngns are commonly used in the mesencephalon for determination of the GABAergic versus glutamatergic transmitter phenotype.

Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord.

Postmitotic neural precursors are generated in the ventricular zone (VZ) of the developing neural tube and immediately migrate to the mantle layer (ML) where they differentiate into mature neurons. Although the regulation of neuronal differentiation and migration has extensively been studied, the behavior of the early postmitotic precursors migrating toward the ML is largely unknown. In this study, we have identified Neph3 as a specific marker for early postmitotic neural precursors in the VZ of the developing spinal cord. Analysis of Neph3 localization by immunofluorescence and immunoelectron microscopy revealed that early neural precursors in the VZ possessed not only pia-connected processes but also ones that reached the ventricle. This apical extension of processes was confirmed by analyzing another early postmitotic marker, Dll1 mRNA, which was actively transported toward the ventricle and accumulated at the termini of the processes. Furthermore, adherens junctions (AJs) were formed around the apical end of processes extending from Neph3- and Dll1 mRNA-positive postmitotic precursors. Taken together, these observations suggest that migrating early postmitotic neural precursors in the VZ of the developing spinal cord form a neuroepithelial cell-like bipolar morphology and communicate with their neighboring cells through AJs.