Jiankai Luo

Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.

Several cadherins are expressed in parasagittal Purkinje cell domains, which can be defined by their afferent and efferent connectivity in the developing and mature cerebellum. By in vivo electroporation in chicken embryos, we demonstrate that Purkinje cell progenitors, which overexpress cadherin-6B or cadherin-7, distribute preferentially to those Purkinje cell domains, which express the respective cadherin endogenously. This differential distribution may be based, at least in part, on the guidance of migrating neurons along neurites that express the same cadherin. Selective induction of apoptosis and cadherin-based cell sorting within cortical domains do not seem to contribute to the differential distribution. These results show that cadherins can tell early neurons where to integrate in functional brain gray matter, possibly by a cadherin-based homotypic adhesive mechanism.

MOLECULAR PROFILING INDICATES AVIAN BRANCHIOMOTOR NUCLEI INVADE THE HINDBRAIN ALAR PLATE

It is generally believed that the spinal cord and hindbrain consist of a motor basal plate and a sensory alar plate. We now have molecular markers for these territories. The relationship of migrating branchiomotor neurons to molecularly defined alar and basal domains was examined in the chicken embryo by mapping the expression of cadherin-7 and cadherin-6B, in comparison to genetic markers for ventrodorsal patterning (Otp, Pax6, Pax7, Nkx2.2, and Shh) and motoneuron subpopulations (Phox2b and Isl1). We show cadherin-7 is expressed in a complete radial domain occupying a lateral region of the hindbrain basal plate. The cadherin-7 domain abuts the medial border of Pax7 expression; this common limit defines, or at least approximates, the basal/alar boundary. The hindbrain branchiomotor neurons originate in the medial part of the basal plate, close to the floor plate. Their cadherin-7-positive axons grow into the alar plate and exit the hindbrain close to the corresponding afferent nerve root. The cadherin-7-positive neuronal cell bodies later translocate laterally, following this axonal trajectory, thereby passing through the cadherin-7-positive basal plate domain. Finally, the cell bodies traverse the molecularly defined basal/alar boundary and move into positions within the alar plate. After the migration has ended, the branchiomotor neurons switch expression from cadherin-7 to cadherin-6B. These findings demonstrate that a specific subset of primary motor neurons, the branchiomotor neurons, migrate into the alar plate of the chicken embryo. Consequently, the century-old concept that all primary motor neurons come to reside in the basal plate should be revised.

Quantitative and kinetic profile of Wnt/β-catenin signaling components during human neural progenitor cell differentiation

ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/β-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/β-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized β-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and β-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/β-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/β-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.

Differentiation of human neural progenitor cells regulated by Wnt-3a.

Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and β-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized β-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/β-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized β-catenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/β-catenin pathway in ReNcell VM cells.

Differential expression of five members of the ADAM family in the developing chicken brain.

ADAMs (a disintegrin and metalloprotease) are a family of trans-membrane multi-domain metalloproteases with multiple functions. So far, more than 35 ADAM family members have been identified from mammalian and nonmammalian sources. Although some functions of ADAMs have been elucidated, their expression patterns remain poorly investigated, especially during CNS development. Here, we cloned the open reading frames or full-length cDNAs of ADAM9, ADAM10, ADAM12, ADAM22 and ADAM23 from chicken embryonic brain, analyzed their evolutionary relationship, and mapped their expression in the embryonic chicken brain by in situ hybridization for the first time. In general, each of the five ADAMs shows a spatially restricted and temporally regulated expression profile. However, the types of tissues and cells, which express each of the five ADAMs, differ from each other. ADAM9 is predominantly expressed in the choroid plexus and in the ventricular layer. ADAM10 is expressed by developing blood vessels, oligodendrocytes, and subsets of neurons and brain nuclei. ADAM12 is expressed by very few brain nuclei, cerebellar Purkinje cells, restricted regions of the neuroepithelium, and some neurons in the deep tectal layers. ADAM22 expression is strong in some brain nuclei and in the pineal gland. ADAM23 is expressed by most gray matter regions and the choroid plexus. The differential expression patterns suggest that the five ADAMs play multiple and versatile roles during brain development.

Ex ovo electroporation for gene transfer into older chicken embryos

In ovo electroporation is an excellent method to ectopically induce or inhibit gene expression in chicken embryos and to study the in vivo function of genes during embryonic development. However, the application of electroporation in ovo to date is limited to an early stage of incubation (< stage 20). In older embryos (> stage 22), the vitelline and allantoic vessels have developed extensively and the in ovo manipulation of the embryo becomes exceedingly difficult. Therefore, in this study, we validate an ex ovo electroporation system, by which the time for performing electroporation can be extended up to at least day 7 of incubation. The application of this method will help to study gene function and regulation at later stages of development in the living chicken embryo. Developmental Dynamics 233:1470–1477, 2005.

Cadherin expression in the developing chicken cochlea

In this study, we demonstrate that eight classic cadherins are differentially expressed in distinct anatomical regions of the cochlea during late stages of chicken embryonic development. Cadherin‐6B is expressed in hair cells and spindle‐shaped cells, while cadherin‐8 mRNA is found only in supporting cells. Cadherin‐11 is widely expressed not only in mesenchymal cell around the cochlea, but also in supporting cells and homogene cells. N‐cadherin is found in the sensory epithelium, the neurons of the acoustic ganglion and on their neurites that target the hair cells. Three closely related cadherins (cadherin‐7, cadherin‐19, and cadherin‐20) are expressed in a partially complementary manner in spindle‐shaped cells and acoustic ganglion cells. R‐cadherin is observed in homogene cells, acoustic ganglion cells, and their projections to hair cells. The expression of classic cadherins in the developing cochlea suggests a role for cadherins in the development of the cochlea. Developmental Dynamics 236:2331–2337, 2007.

Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells

BackgroundHypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process.ResultsIn this study we demonstrate that differentiation of human fetal neural progenitor cells under hypoxic conditions results in an increased neurogenesis. In addition, expansion and proliferation under lowered oxygen conditions also increased neuronal differentiation, although proliferation rates were not altered compared to normoxic conditions. Erythropoietin partially mimicked these hypoxic effects, as shown by an increase of the metabolic activity during differentiation and protection of differentiated cells from apoptosis.ConclusionThese results provide evidence that hypoxia promotes the differentiation of human fetal neural progenitor cells, and identifies the involvement of erythropoietin during differentiation as well as different cellular mechanisms underlying the induction of differentiation mediated by lowered oxygen levels.

Cadherin-19 expression is restricted to myelin-forming cells in the chicken embryo

We cloned chicken cadherin-19 that demonstrates high similarity to human and rat cadherin-19. Chicken cadherin-19 is a type II classic cadherin that is located on the long arm of chicken chromosome 2 and is composed of 13 exons and 12 introns. The expression profile of cadherin-19 was analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization during chicken embryonic development. Its expression starts at E2.5, then gradually increases to reach a peak at E20. In contrast to previous results obtained in rat, chicken cadherin-19 is expressed both in Schwann cells and oligodendrocytes, also at late stages of development. We found no other cell type positive for cadherin-19 in the chicken embryo throughout development, suggesting that cadherin-19 is selectively expressed by myelin-forming cells and might play a role in myelin formation. The sequence of cadherin-19 shares high similarity with that of cadherin-7 and cadherin-20, and the three genes form a cluster on chromosome 2. Their expression patterns, however, are rather distinct although partial overlap is observed. For example, cadherin-19 and cadherin-7 are co-expressed by Schwann cells but not by oligodendrocytes. Moreover, a subset of interneurons express cadherin-7 but not cadherin-19 or cadherin-20. Despite their close genetic relation, the three cadherins have acquired functions in rather different cell types during nervous system development.

Expression of seven members of the ADAM family in developing chicken spinal cord

The expression patterns of seven members of the ADAM (a disintegrin and metalloprotease) family, including ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23, were analyzed in the developing chicken lumbar spinal cord by in situ hybridization and immunohistochemistry. Results show that each individual ADAM is expressed and regulated spatiotemporally in the lumbar cord and its surrounding tissues. ADAM9, ADAM10, ADAM22, and ADAM23 are expressed predominantly by motoneurons in the motor column and by sensory neurons in the dorsal root ganglia, each with a different expression pattern. ADAM12 and ADAM13 are mainly expressed in the meninges around the lumbar cord and in the condensed sheets of chondroblasts around the vertebrae. ADAM17 expression is strong in the ventricular layer and limited to early stages. The differential expression of the ADAMs in the lumbar cord suggests that the ADAMs play a regulatory role in development of the spinal cord. Developmental Dynamics 239:1246–1254, 2010.