Ciqing Yang

N-cadherin regulates beta-catenin signal and its misexpression perturbs commissural axon projection in the developing chicken spinal cord

N-cadherin is a calcium-sensitive cell adhesion molecule that plays an important role in the formation of the neural circuit and the development of the nervous system. In the present study, we investigated the function of N-cadherin in cell–cell connection in vitro with HEK293T cells, and in commissural axon projections in the developing chicken spinal cord using in ovo electroporation. Cell–cell connections increased with N-cadherin overexpression in HEK293T cells, while cell contacts disappeared after co-transfection with an N-cadherin-shRNA plasmid. The knockdown of N-cadherin caused the accumulation of β-catenin in the nucleus, supporting the notion that N-cadherin regulates β-catenin signaling in vitro. Furthermore, N-cadherin misexpression perturbed commissural axon projections in the spinal cord. The overexpression of N-cadherin reduced the number of axons that projected alongside the contralateral margin of the floor plate, and formed intermediate longitudinal commissural axons. In contrast, the knockdown of N-cadherin perturbed commissural axon projections significantly, affecting the projections alongside the contralateral margin of the floor plate, but did not affect intermediate longitudinal commissural axons. Taken together, these findings suggest that N-cadherin regulates commissural axon projections in the developing chicken spinal cord.

Sonic Hedgehog Regulation of the Neural Precursor Cell Fate During Chicken Optic Tectum Development

During nervous system development, neurons project axons over long distances to reach the appropriate targets for correct neural circuit formation. Sonic hedgehog (Shh) is a secreted protein and plays a key role in regulating vertebrate embryogenesis, especially in central nervous system (CNS) patterning, including neuronal migration and axonal projection in the brain and spinal cord. In the developing ventral midbrain, Shh is sufficient to specify a striped pattern of cell fates. Little is known about the molecular mechanisms underlying the Shh regulation of the neural precursor cell fate during the optic tectum development. Here, we aimed at studying how Shh might regulate chicken optic tectum patterning. In the present study, in ovo electroporation methods were employed to achieve the overexpression of Shh in the optic tectum during chicken embryo development. Besides, the study combined in ovo electroporation and neuron isolation culturing to study the function of Shh in vivo and in vitro. The fluorescent immunohistochemistry methods were used to check the related indicators. The results showed that Shh overexpression caused 87.8% of cells to be distributed to the stratum griseum central (SGC) layer, while only 39.3% of the GFP-transfected cells resided in the SGC layer in the control group. Shh overexpression also reduced the axon length in vivo and in vitro. In conclusion, we provide evidence that Shh regulates the neural precursor cell fate during chicken optic tectum development. Shh overexpression impairs neuronal migration and may affect the fate determination of transfected neurons.

N-Cadherin Upregulation Promotes the Neurogenic Differentiation of Menstrual Blood-Derived Endometrial Stem Cells

Peripheral nerve injuries are typically caused by either trauma or medical disorders, and recently, stem cell-based therapies have provided a promising treatment approach. Menstrual blood-derived endometrial stem cells (MenSCs) are considered an ideal therapeutic option for peripheral nerve repair due to a noninvasive collection procedure and their high proliferation rate and immunological tolerance. Here, we successfully isolated MenSCs and examined their biological characteristics including their morphology, multipotency, and immunophenotype. Subsequent in vitro studies demonstrated that MenSCs express high levels of neurotrophic factors, such as NT3, NT4, BDNF, and NGF, and are capable of transdifferentiating into glial-like cells under conventional induction conditions. Moreover, upregulation of N-cadherin (N-cad) mRNA and protein expression was observed after neurogenic differentiation. In vivo studies clearly showed that N-cad knockdown via in utero electroporation perturbed the migration and maturation of mouse neural precursor cells (NPCs). Finally, a further transfection assay also confirmed that N-cad upregulation in MenSCs results in the expression of S100. Collectively, our results confirmed the paracrine effect of MenSCs on neuroprotection as well as their potential for transdifferentiation into glial-like cells and demonstrated that N-cad upregulation promotes the neurogenic differentiation of MenSCs, thereby providing support for transgenic MenSC-based therapy for peripheral nerve injury.

Wnt3a Ectopic Expression Interferes Axonal Projection and Motor Neuron Positioning During the Chicken Spinal Cord Development

The formation of dorsal-ventral axis of the spinal cord is controlled largely by dorsal signals such as Wnts (which are members of the wingless + MMTV integrants, Int family), besides ventral signals such as sonic hedgehog (Shh). Wnt3a, one of the Wnt family members, is involved in multiple cellular functions, including self-renewal, proliferation, differentiation, and motility. Here, we aim to study the mechanism of the regulation of chicken spinal cord patterning by Wnt3a. In this study, Wnt3a was ectopically expressed in the spinal cord of developing chicken embryos by in ovo electroporation. The results of immunofluorescent staining revealed that Wnt3a ectopic expression caused the abnormality of commissural axonal projection and the formation of nerve fibers was interrupted. It is worth noting that neurons in the ventricular zone, especially motor neurons, could not migrate laterally after the Wnt3a overexpression, which led to the malformation of motor column. In addition, we found that neurons could not protrude axons outwardly after overexpression of Wnt3a in the spinal cord. It was also found that Wnt3a overexpression inhibited the outgrowth of processes in culturing SH-SY5Y cells. In conclusion, we proposed that Wnt3a regulates neuronal morphology, which subsequently disrupts axonal projection and motor neuron positioning during spinal cord development.

Effects of N-cadherin on neuronal migration during chicken optic tectum development

N-cadherin, a member of the cadherin family, plays an important role in neural development. In addition, N-cadherin has been reported to be crucial in neuronal migration, axonal outgrowth, and axonal path-finding. However, the mechanism underlying the effects of N-cadherin in neuronal migration is not entirely clear. In this study, we investigated the overexpression or knockdown of N-cadherin in the optic tectum during chicken embryo development, and then analyzed the effect of N-cadherin on neuronal migration. The results showed that compared with the control group, in the N-cadherin knockdown group, the neuronal migration of the optic tectum was significantly affected and could not arrive at destination. The stratum griseum central layer of the optic tectum mainly includes multipolar neurons, which could not be formed after the knockdown of N-cadherin, and more neurons form the bipolar or monopolar neurons compared with the control group. Compared with the control group, more cells stayed in the neuroepithelium layer. The axonal length in the optic tectum was significantly (P < 0.001) shorter in the N-cadherin knockdown group than in the control group. These results reveal that the knockdown of N-cadherin mainly affects the length of axons and formation of multipolar neurons in the development of the chicken optic tectum, which eventually results in the inhibition of neuronal migration.

The effect of sonic hedgehog on motor neuron positioning in the spinal cord during chicken embryo development

Sonic hedgehog (Shh) is a vertebrate homologue of the secreted Drosophila protein hedgehog, and is expressed by the notochord and the floor plate in the developing spinal cord. Shh provides signals relevant for positional information, cell proliferation, and possibly cell survival depending on the time and location of the expression. Although the role of Shh in providing positional information in the neural tube has been experimentally proven, the exact underlying mechanism still remains unclear. In this study, we report that overexpression of Shh affects motor neuron positioning in the spinal cord during chicken embryo development by inducing abnormalities in the structure of the motor column and motor neuron integration. In addition, Shh overexpression inhibits the expression of dorsal transcription factors and commissural axon projections. Our results indicate that correct location of Shh expression is the key to the formation of the motor column. In conclusion, the overexpression of Shh in the spinal cord not only affects the positioning of motor neurons, but also induces abnormalities in the structure of the motor column.

Direction of commissural axon projections in different regions of the spinal cord during chicken embryonic development

Few researchers have investigated the direction of commissural axon projections on the contralateral side of the vertebrate embryonic spinal cord, especially for comparison between its different regions. In this study, pCAGGS-GFP plasmid expression was limited to different regions of the chicken embryonic spinal cord (cervical, anterior limb, anterior thorax, posterior thorax and posterior limb) at E3 using in ovo electroporation with modified electrodes and optimal electroporation conditions. Then open-book technique was performed at E6 to analyze the direction of axon projections in different spinal cord regions. The results show that in the five investigated regions, most axons projected rostrally after crossing the floor plate while a minority projected caudally. And there was a significant difference between the rostral and caudal projection quantities (P<0.01). The ratio of rostral and caudal projections was significantly different between the five investigated regions (P<0.05), except between the cervical region and the anterior limb (P>0.05). The projections were most likely to be rostral for the posterior limb followed by the posterior thorax, cervical region, anterior limb and anterior thorax. Our data for the direction of the commissural axon projections will be helpful in the future analyses of axon projection mechanisms and spinal cord-brain circuit formation.

Telocytes: a potential defender in the spleen of Npc1 mutant mice

Niemann–Pick disease, type C1 (Npc1), is an atypical lysosomal storage disorder caused by autosomal recessive inheritance of mutations in Npc1 gene. In the Npc1 mutant mice (Npc1−/−), the initial manifestation is enlarged spleen, concomitant with free cholesterol accumulation. Telocytes (TCs), a novel type of interstitial cell, exist in a variety of tissues including spleen, presumably thought to be involved in many biological processes such as nursing stem cells and recruiting inflammatory cells. In this study, we found that the spleen is significantly enlarged in Npc1−/− mice, and the results from transmission electron microscopy examination and immunostaining using three different TCs markers, c‐Kit, CD34 and Vimentin revealed significantly increased splenic TCs in Npc1−/− mice. Furthermore, hematopoietic stem cells and macrophages were also elevated in Npc1−/− spleen. Taken together, our data indicate that splenic TCs might alleviate the progress of splenic malfunction via recruiting hematopoietic stem cells and macrophages.