人胚不同脑区神经前体细胞的分离培养及分化特性的比较

目的研究人胚不同脑区神经前体细胞(neural progenitor cells,NPCs)培养及增殖分化特性。方法取14-17周人胚脑区组织,分为新皮质、纹状体、间脑、中脑、后脑和延髓组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各脑区培养细胞的生长、增殖状况。新皮质、纹状体及间脑来源的神经球分化后,运用免疫荧光细胞化学法比较神经元及星形胶质细胞的比例。结果各脑区培养出的悬浮细胞球巢蛋白抗原阳性,可分化为MAP2或GFAP阳性细胞,且BrdU掺入实验阳性。体外培养第3d,纹状体及间脑组均可见大量神经球,且纹状体组明显多于间脑组;新皮质组传代后可见较多神经球;其它组仅见个别神经球。新皮质、纹状体、间脑来源的NPCs诱导分化后,MAP2或GFAP阳性细胞率各组间比较差异无显著性。结论人胚不同脑区均可培养出NPCs,从易到难依次为纹状体、间脑、新皮质及其它脑区。新皮质、纹状体、间脑来源的NPCs体外分化比例一致。     

Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, -, - and -catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.     

Pluripotentiality of the 2-day-old avian germinative neuroepithelium

In a previous study using chick/quail chimeric embryos with homotopic transplants (Martinez & Alvarado-Mallart, 1989b), we have delimited in the 2-day-old avian embryo the areas of the neural tube giving rise to optic tectum and mesencephalic grissea as well as to isthmic grissea and cerebellum: respectively, "mesencephalic" and "metencephalic" alar plates. To investigate the determination or the competence of these areas, portions of these germinative neuroepithelia from a quail embryo were transplanted in substitution for other areas of the chick neural tube. The analysis of the chimeric brains was done by comparing alternating transverse sections stained for cytoarchitecture and with two different techniques to recognize transplanted versus host cells: either the Feulgen and Rossenbeck DNA histochemical reaction and/or immunohistochemical methods with a monoclonal antibody recognizing quail but not chick cells. The eventual visual innervation of the quail graft was analyzed in many cases by injecting anterograde axonal tracers in the eye contralateral to the graft. The results are as follows: (1) caudal metencephalon transferred to mesencephalon maintained in all cases its presumptive cerebellar phenotype, whereas (2) rostral metencephalon transferred to mesencephalon changed its fate to a tectal phenotype but maintained its cerebellar fate when transferred to diencephalon; (3) caudal mesencephalon maintained its tectal fate in 65% of the cases when transferred to diencephalon, whereas (4) rostral mesencephalon transferred to a cerebellar domain changed its fate and became influenced by the surrounding structures in all cases, but only in 85% of the cases when it was transplanted to diencephalon; (5) the in situ host diencephalon, isolated from its normal environment by a mesencephalic graft, is competent to change its fate and express a mesencephalic phenotype. These results demonstrate that at least some regions of the germinative neuroepithelium from either metencephalon, mesencephalon, and diencephalon are still pluripotent in the 2-day-old avian embryo and that their fate seems to be under the influence of the surrounding structures. Rostral mesencephalon and rostral metencephalon have been more easily influenced by environmental factors than their caudal counterparts, suggesting that regions providing instructive positional factors exist within the 2-day-old germinative neuroepithelium. These regions might play an important role in the determination of the various segments of the neural tube.     

Cortex-specific distribution of membrane-bound factors that promote neurite outgrowth of mitral cells in culture

To analyze regional differences in the embryonic mouse brain with respect to environmental influence on mitral cell neurites, olfactory bulb fragments were cultured on layers of brain cells which had been dissociated from various regions. Long mitral cell neurites elongated on paleocortex and neocortex cell layers, but not on the septum, mesencephalon, or diencephalon cell layers. Cell membranes prepared from the paleocortex and neocortex also supported outgrowth of long mitral cell neurites, but cell membranes prepared from the septum, mesencephalon, or diencephalon did not. The supportability of mitral cell neurites in the paleocortex and neocortex membranes was completely abolished by trypsin treatment. Neurite outgrowth of the mitral cells on poly-L -lysine was not inhibited by the mesencephalon or diencephalon membranes, but was promoted by the paleocortex and neocortex membranes. These results indicate that the paleocortex and neocortex regions selectively express membrane-bound factors which promote neurite outgrowth of mitral cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 415–425, 1997.     

PCP4 is highly expressed in ectoderm and particularly in neuroectoderm derivatives during mouse embryogenesis

PCP4 (PEP-19) belongs to a family of proteins involved in calcium transduction signals and binds calmodulin via an IQ motif, in a calcium independent manner. PCP4 gene maps to murine chromosome 16 and in human to chromosome 21. Murine PCP4 expression in the brain has been detected by Northern blot analysis to be mainly post-natal and in the adult to have a neuronal pattern. To investigate if it might have a role earlier in development, we analyzed its expression during mouse embryogenesis by in situ hybridization from E7.5 post-coitum (p.c.) to E17.5 p.c., and in P0 brain. Early, at E7.5, a high expression is restricted to the extra embryonic ectoderm. Embryonic expression starts at E9.5. At E10.5, PCP4 shows a strong signal in the post-mitotic cells of the diencephalon, the metencephalon and the myelencephalon and in the dorsal and cranial ganglia. The floor plate is also densely labelled. At E17.5, PCP4 is expressed in the central nervous system, in the myenteric plexus, and in other ectoderm derivatives, for instance the lens, the hairy cells of the cochlea, the enamel organ and the hair follicles. Thus, during embryogenesis PCP4 is mainly expressed in ectoderm and neuroectoderm comprising neural crest derived cells.     

Crossregulation between En-2 and Wnt-1 in chick tectal development

En-1, En-2 and Wnt-1 are proposed to be essential signals for the development of the optic tectum in chick embryos. Drosophila engrailed and wingless , homologs of En ( En-1 and En-2 ) and Wnt-1 , respectively, have been shown to crossregulate each other. In the present paper, it is reported that crossregulation between En-2 and Wnt-1 is preserved in the development of the chick optic tectum. When En-2 is overexpressed by the replication competent retroviral vector, Wnt-1 is expressed ectopically at the dorsal midline of the diencephalon. When Wnt-1 is introduced extrinsically either by ectopic transplantation of mesencephalon, or by implantation of Wnt-1 producing cells, En-2 is induced ectopically at the dorsal midline of the tel-diencephalic border. Thus, ectopic expression of En-2 and Wnt-1 leads to crossregulation of each other in the chick brain. As diencephalon transdifferentiates into the optic tectum by an appropriate signal, the crossregulation of En-2 and Wnt-1 in the diencephalon may mimic the relationship required for early development in the tectum.     

Inductive signal and tissue responsiveness defining the tectum and the cerebellum

The mes/metencephalic boundary (isthmus) has an organizing activity for mesencephalon and metencephalon. The candidate signaling molecule is Fgf8 whose mRNA is localized in the region where the cerebellum differentiates. Responding to this signal, the cerebellum differentiates in the metencephalon and the tectum differentiates in the mesencephalon. Based on the assumption that strong Fgf8 signal induces the cerebellum and that the Fgf8b signal is stronger than that of Fgf8a, we carried out experiments to misexpress Fgf8b and Fgf8a in chick embryos. Fgf8a did not affect the expression pattern of Otx2, Gbx2 or Irx2. En2 expression was upregulated in the mesencephalon and in the diencephalon by Fgf8a. Consequently, Fgf8a misexpression resulted in the transformation of the presumptive diencephalon to the fate of the mesencephalon. In contrast, Fgf8b repressed Otx2 expression, but upregulated Gbx2 and Irx2 expression in the mesencephalon. As a result, Fgf8b completely changed the fate of the mesencephalic alar plate to cerebellum. Quantitative analysis showed that Fgf8b signal is 100 times stronger than Fgf8a signal. Co-transfection of Fgf8b with Otx2 indicates that Otx2 is a key molecule in mesencephalic generation. We have shown by RT-PCR that both Fgf8a and Fgf8b are expressed, Fgf8b expression prevailing in the isthmic region. The results all support our working hypothesis that the strong Fgf8 signal induces the neural tissue around the isthmus to differentiate into the cerebellum.     

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Delta(5)P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450(C17)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(C17)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(C17)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including 17OH-Delta(5)P, 17OH-P, DHEA and Delta(4). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450(C17) inhibitor. This is the first detailed immunohistochemical mapping of P450(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.     

Regulation of Otx2 expression and its functions in mouse forebrain and midbrain

Otx2 expression in the forebrain and midbrain was found to be regulated by two distinct enhancers (FM and FM2) located at 75 kb 5' upstream and 115 kb 3' downstream. The activities of these two enhancers were absent in anterior neuroectoderm earlier than E8.0; however, at E9.5 their regions of activity spanned the entire mesencephalon and diencephalon with their caudal limits at the boundary with the metencephalon or isthmus. In telencephalon, activities were found only in the dorsomedial aspect. Potential binding sites of OTX and TCF were essential to FM activity, and TCF sites were also essential to FM2 activity. The FM2 enhancer appears to be unique to rodent; however, the FM enhancer region is deeply conserved in gnathostomes. Studies of mutants lacking FM or FM2 enhancer demonstrated that these enhancers indeed regulate Otx2 expression in forebrain and midbrain. Development of mesencephalic and diencephalic regions was differentially regulated in a dose-dependent manner by the cooperation between Otx1 and Otx2 under FM and FM2 enhancers: the more caudal the structure the higher the OTX dose requirement. At E10.5 Otx1-/-Otx2DeltaFM/DeltaFM mutants, in which Otx2 expression under the FM2 enhancer remained, exhibited almost complete loss of the entire diencephalon and mesencephalon; the telencephalon did, however, develop.     

The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum.

Mice homozygous for null alleles of the putative signaling molecule Wnt-1 have a reproducible phenotype: loss of the midbrain and adjacent cerebellar component of the metencephalon. By examining embryonic expression of the mouse engrailed (En) genes, from 8.0 to 9.5 days postcoitum, we demonstrate that Wnt-1 primarily regulates midbrain development. The midbrain itself is required for normal development of the metencephalon. Thus, the observed neonatal phenotype is explained by a series of early events, within 48 hr of neural plate induction, that leads to a complete loss of En domains in the anterior central nervous system. Wnt-1 and a related gene, Wnt-3a, are coexpressed from early somite stages in dorsal aspects of the myelencephalon and spinal cord. We suggest that functional redundancy between these two genes accounts for the lack of a caudal central nervous system phenotype.     

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.     

Differential responses of temporal and nasal retinal neurites to regional-specific cues in the mouse retinofugal pathway

Retinal explants of mouse embryos were cultured together with explants of different regions in the retinofugal pathway in order to investigate whether ventral temporal (VT) and dorsal nasal (DN) retinal neurites showed differential responses to regional-specific cues in the pathway. In the presence of the chiasm, biased outgrowth of retinal neurites was found in explants of both retinal regions, which was accompanied by a reduction in total neurite growth in the VT but not the DN retina. Such differential responses to the diffusible negative influence were also observed when explants of two retinal origins were cocultured with the ventral diencephalon, but were not found with the dorsal diencephalon that contains targets of the optic axons. Indeed, extensive neurite invasion was found in the dorsal diencephalic explants and this ingrowth was more prominent for VT than DN neurites, showing a difference in axons from a distinct position in the retina to contact-mediated stimulatory activity within the target nuclei. We conclude that neurites from different regions of the retina show differential responses to the regional-specific cues in the diencephalon. These cues exist in both diffusible and contact-mediated forms that may shape the characteristic course and organization of retinal axons in decision regions of the optic pathway and the visual targets.     

Role of Pax3/7 in the tectum regionalization

Pax3/7 is expressed in the alar plate of the mesencephalon. The optic tectum differentiates from the alar plate of the mesencephalon, and expression of Pax3/7 is well correlated to the tectum development. To explore the function of Pax3 and Pax7 in the tectum development, we misexpressed Pax3 and Pax7 in the diencephalon and ventral mesencephalon. Morphological and molecular marker gene analysis indicated that Pax3 and Pax7 misexpression caused fate change of the alar plate of the presumptive diencephalon to that of the mesencephalon, that is, a tectum and a torus semicircularis were formed ectopically. Ectopic tectum in the diencephalon appeared to be generated through sequential induction of Fgf8, En2 and Pax3/7. In ventral mesencephalon, which expresses En but does not differentiate to the tectum in normal development, Pax3 and Pax7 misexpression induced ectopic tectum. In normal development, Pax3 and Pax7 expression in the mesencephalon commences after Otx2, En and Pax2/5 expression. In addition, expression domain of Pax3 and Pax7 is well consistent with presumptive tectum region in a dorsoventral axis. Taken together with normal expression pattern of Pax3 and Pax7, results of misexpression experiments suggest that Pax3 and Pax7 define the tectum region subsequent to the function of Otx2 and En.     

E-cadherin expression in a particular subset of sensory neurons.

We found that the dorsal root ganglia (DRG) and trigeminal ganglia of mouse embryos express the E-cadherin cell-cell adhesion molecule and analyzed its expression profile. E-cadherin expression began around Embryonic Day 12 (E12) in these ganglia, thereafter increased, and persisted to the adult stage. This cadherin was expressed by 10 and 30% of DRG neurons in E17 and postnatal animals, respectively, as well as by satellite cells and some Schwann cells. E-cadherin-positive primary sensory fibers terminated only in a narrow region of the dorsal horn of the spinal cord, which was identified as part of lamina II by double-staining for E-cadherin and substance P or somatostatin. This E-cadherin expressing area of the spinal cord extended to part of the trigeminal nucleus in the medulla. These results showed that E-cadherin is expressed in a particular subset of primary sensory neurons which may have specific functional properties. We suggest that this adhesion molecule may play a role in the selective adhesion of sensory neuronal fibers.     

Effects of acidic and basic fibroblast growth factors (aFGF, bFGF) on glial precursor cell proliferation: age dependency and brain region specificity.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are present in high levels in most areas of the embryonic rodent brain. To begin to understand the role of these growth factors in brain development, the effects of aFGF and bFGF on dissociated cell cultures prepared from embryonic and neonatal rat brain were studied. Addition of aFGF and heparin or bFGF alone to serum-free cultures of the dissociated Embryonic Day (E) 14.5 mesencephalon stimulates cell proliferation, as judged by [3H]thymidine autoradiography, leading to a maximal 75-fold increase in the total number of cells. This effect is dose-dependent with half-maximal increases at concentrations of about 5-6 ng/ml of aFGF or bFGF and is inhibited by the FGF antagonist HBGF-1U. The effect of aFGF on cell proliferation in cultures prepared from E14.5 mesencephalon is similar to that in cultures prepared from E14.5 cortex. However, in cultures prepared from E14.5 rhombencephalon or diencephalon, the proliferative effect of aFGF is much reduced. In all brain areas studied, the proliferative effect of aFGF declines with increasing age. Immunocytochemical analysis of E14.5 mesencephalic cultures demonstrated that the aFGF-induced increase in cell number is due to the proliferation of A2B5-immunoreactive (IR) glial precursor cells, but not of neuronal precursors, fibroblasts, or microglial cells. Moreover, differentiated glial fibrillary acidic protein-IR astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase-IR oligodendrocytes were not observed in cultures continuously treated with aFGF or bFGF, but were observed in high numbers after removal of the growth factors. These results suggest (1) that aFGF and bFGF are potent mitogens for glial precursor cells in all embryonic brain regions, (2) that the magnitude of the effects of aFGF depends on embryonic age and brain region, and (3) that both growth factors inhibit the differentiation of astrocyte or oligodendrocyte precursors. These observations made in vitro strongly support the hypothesis that FGF plays a critical role in gliogenesis and the timing of glial differentiation in the brain.