小鼠视网膜片层化及神经干细胞的增殖与分化

目的 通过观察小鼠视网膜神经干细胞增殖与双极细胞分化过程,研究视网膜的发生及片层化。方法 应用免疫荧光、5’-溴脱氧尿嘧啶核苷（BrdU）检测技术和HE染色法对胚胎及出生后小鼠视网膜形态结构及神经干细胞的增殖、分化进行观察,对视网膜BrdU和蛋白激酶Cα(PKC-α)阳性细胞密度进行统计。结果 1.小鼠视网膜在胚胎时期分化出色素上皮层、神经母细胞层和神经节细胞层。出生后,神经母细胞层逐渐分化出各个层,至小鼠睁眼时基本分化完全。2.小鼠视网膜干细胞在胚胎期大量增殖,出生后增殖放慢并逐渐分化为各类细胞。经统计分析发现,视网膜干细胞在胚胎时期数量逐渐增多,到出生当天数量达到最大值,出生后,神经干细胞开始分化,数量逐渐减少。3.小鼠视网膜双极细胞从出生后第5天（P5）开始发育,至P20时发育完全。结论 小鼠视网膜的片层化与其功能的成熟相一致,视网膜的神经干细胞在出生后前期为分化高峰期,逐渐分化为不同类型的细胞。P10以后仅在睫状体处存在神经干细胞,可能与成年以后的修复功能相关。     

Cell intrinsic mechanisms regulate mouse cerebellar granule neuron differentiation

Cerebellar granule cells isolated from postnatal day 7 mice, and cultured in minimal medium containing only insulin-like growth factor-I (IGF-I), both survive and differentiate. This differentiation is marked by neurite growth and expression of genes associated with terminal differentiation, the myocyte-specific enhancer factor 2A (MEF2A) and the α6 subunit of the γ-aminobutyric acid_A receptor (GABA_Aα6). Percoll gradient purified granule cells maintained without IGF-I, in minimal medium alone or in medium containing the antioxidant N-acetylcysteine (NAC), also express MEF2A and GABA_Aα6. Thus, cultured granule neurons can differentiate to some extent cell-autonomously and IGF-I may not be a critical factor for this process.     

小鼠视网膜神经免疫系统的组织发生

目的 探讨小鼠视网膜神经免疫系统中小胶质细胞和血视网膜屏障（BRB）的发育过程,及视网膜神经免疫系统的组织发生。方法 选取不同年龄点的昆明小鼠各5～10只,应用免疫荧光染色、DiI散射标记、明胶墨汁灌注和透射电子显微镜技术,对视网膜上小胶质细胞和BRB的发育进行研究。 结果 在孕10 d（E10）时,视网膜上就已经出现了小胶质细胞,并且均匀分布于整个视网膜,随着发育小胶质细胞的形态由阿米巴样变成分支状。出生后小胶质细胞数量不断增多,在出生5 d（P5）时达到最大值,之后细胞数量有所下降,P30后趋于稳定。视网膜上血管的发生是在出生后由视乳头开始呈辐射状向四周扩散的,在P10左右浅层血管网覆盖整个视网膜,之后不断向下延伸形成深层血管网。随着年龄增长,血管体密度呈下降趋势。BRB在P30时发育成熟,主要由管腔光滑的内皮细胞、厚度均一的基底膜、薄层星形胶质细胞的终足和周细胞构成。 结论 小胶质细胞随着发育变得更加成熟,数量变化呈抛物线状;P30时,BRB的各个组成部分已发育完善,各结构之间关系密切。视网膜神经免疫系统的重要组成部分--小胶质细胞和BRB,具有一定的抗感染能力,能够有效地抵抗病原菌的感染。     

Mash1 promotes neuronal differentiation in the retina

Background: Mash1, a mammalian homologue of Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurones. Whereas Mash1 is expressed during retinal development, no apparent abnormalities were found during embryogenesis as well as at birth in Mash1-null retina, suggesting that early differentiating cells such as ganglion, amacrine and cone cells develop normally. Because Mash1-null mice die soon after birth, their postnatal development cannot be examined in vivo. Thus, it remains to be determined whether or not Mash1 functions in postnatal development of retina. Results: Here, Mash1 roles in postnatal development of retina was examined by using retinal explant that develops like in vivo retina. Without Mash1, differentiation of late appearing cells such as rod, horizontal, and bipolar cells was delayed and the final number of bipolar cells was significantly reduced. In contrast, vimentin-positive cells (probably Müller glial cells) were increased in Mash1-null retina. Conclusions: These results provide evidence that Mash1 promotes neuronal differentiation during retinal development and is essential for proper ratios of retinal cell types.     

ON direction-selective ganglion cells in the mouse retina

Two types of ganglion cells (RGCs) compute motion direction in the retina: the ON–OFF direction-selective ganglion cells (DSGCs) and the ON DSGCs. The ON DSGCs are much less studied mostly due to the low encounter rate. In this study, we investigated the physiology, dendritic morphology and synaptic inputs of the ON DSGCs in the mouse retina. When a visual stimulus moved back and forth in the preferred–null axis, we found that the ON DSGCs exhibited a larger EPSC when the visual stimulus moved in the preferred direction and a larger IPSC in the opposite, or null direction, similar to what has been found in ON–OFF DSGCs. This similar synaptic input pattern is in contrast to other well-known differences, namely: profile of velocity sensitivity, distribution of preferred directions, and different central projection of the axons. Immunohistochemical staining showed that the dendrites of ON DSGCs exhibited tight cofasciculation with the cholinergic plexus. These findings suggest that cholinergic amacrine cells may play an important role in generating direction selectivity in the ON DSGCs, and that the mechanism for coding motion direction is probably similar for the two types of DSGCs in the retina.     

Localization of primary cilia in mouse retina

The primary cilia are considered as “cellular antennae” that sense and interchange information with the extracellular environment. Nearly all mammalian cells have a single primary cilium. In the retina, the outer segment of the photoreceptor is known to be a specialized form of primary cilium, but studies on cilia in other layers of the retina are scarce. In this study, we investigated the expression of primary cilia in the whole thickness of the mouse retina using immunofluorescence with three different ciliary markers: Arl13b, acetylated α-tubulin and adenylyl cyclase III. Our results show positive reactions in the photoreceptor layer, outer plexiform layer and ganglion cell layer, which might suggest the possible presence of primary cilia in these areas, but we could not directly prove the strand-like shape of cilia in those areas. In the outer plexiform layer, all three markers showed intense staining along the neuronal synapses, which suggests that the neuronal processes themselves might share the features of cilia.     

Physiological clustering of visual channels in the mouse retina

Anatomy predicts that mammalian retinas should have in excess of 12 physiological channels, each encoding a specific aspect of the visual scene. Although several channels have been correlated with morphological cell types, the number of morphological types generally exceeds the known physiological types. Here, we attempted to sort the ganglion cells of the mouse retina purely on a physiological basis. The null hypothesis was that the outputs of the ganglion cells form a continuum or should be divided into only a few types. We recorded the spiking output of 471 retinal ganglion cells on a multielectrode array while presenting 4 classes of visual stimuli. Five parameters were chosen to describe each cell's response characteristics, including relative amplitude of the ON and OFF responses, response latency, response transience, direction selectivity, and the receptive field surround. We compared the results of four clustering routines and judged the results using the relevant validation indices. The optimal partition was the 12-cluster solution of the Fuzzy Gustafson-Kessel algorithm. This classification contained three visual channels that carried predominately OFF responses, six that carried ON responses, and three that carried both ON and OFF information. They differed in other parameters as well. Other evidence suggests that the true number of cell types in the mouse retina may be somewhat larger than 12, and a definitive typology will probably require broader stimulus sets and characterization of more response parameters. Nonetheless, the present results do allow us to reject the null hypothesis: it appears that in addition to well-known cell types (such as the ON-OFF direction selectivity cells) numerous other cell classes can be identified in the mouse retina based solely on their responses to a standard set of simple visual stimuli.     

H3K27me3 demethylase UTX regulates the differentiation of a subset of bipolar cells in the mouse retina

Di‐ and trimethylation of lysine 27 on histone 3 (H3K27me2/3) is a critical gene repression mechanism. We previously showed that down‐regulation of the H3K27 demethylase, Jumonji domain‐containing protein 3 (JMJD3), resulted in a reduced number of protein kinase C (PKC)α‐positive rod ON‐bipolar cells. In this work, we focused on the role of another H3K27 demethylase, ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX), in retinal development. UTX was expressed in the retinal progenitor cells of the embryonic mouse retina and was observed in the inner nuclear layer during late retinal development and in the mature retina. The short hairpin RNA‐mediated knockdown of Utx in a mouse retinal explant led to a reduced number of PKCα‐positive rod ON‐bipolar cells. However, other retinal subtypes were unaffected by this knockdown. Using a retina‐specific knockout of Utx in mice, the in vivo effects of UTX down‐regulation were examined. Again, the number of PKCα‐positive rod ON‐bipolar cells was reduced, and no other apparent phenotypes, including retinal progenitor proliferation, apoptosis or differentiation, were observed. Finally, we examined retina‐specific Utx and Jmjd3 double‐knockout mice and found that although the number of rod ON‐bipolar cells was reduced, no additional effects from the loss of Utx and Jmjd3 were observed. Taken together, our data show that UTX contributes to retinal differentiation in a lineage‐specific manner.     

Expression of the neurokinin 1 receptor in the mouse retina

Previous studies have revealed that the expression pattern of the neurokinin 1 receptor (the preferred receptor for substance P, SP) varies in different mammalian retinas. We investigated NK1 receptor expression in the mouse retina to provide background information for future studies in transgenic mice on SP functional roles in the retina. Mouse retinal sections were treated for single and double-label immunofluorescence. NK1 receptor immunoreactivity was in bipolar cells and in numerous amacrine cells. Double-label studies showed that NK1 receptor-expressing bipolar cells constituted a population of ON-type cone bipolar cells, since they were distinct from rod bipolar cells and contained glycine. They were nonrandomly distributed with highest density in central retina. These cells were similar and may correspond to the population of NK1 receptor-expressing bipolar cells of the rabbit retina. Different subsets of NK1 receptor-expressing amacrine cells were identified on the basis of the expression of selected neurotransmitter substances: i) about 23% of NK1 receptor-expressing amacrine cells also contained glycine; ii) the remaining 77% were likely to be GABAergic, although some inconsistency was observed in the GABA immunostaining obtained with two different GABA antibodies; iii) all dopaminergic amacrine cells also expressed NK1 receptors; iv) about one third of SP-containing amacrine cells also expressed NK1 receptors. These findings confirm and expand previous observations in rat and rabbit retinas. In particular, common to all three species is the expression of NK1 receptors in dopaminergic amacrine cells, indicating that SP neurotransmission may be a universal feature of the circuitry of the dopaminergic amacrine cell. Peculiar to the mouse retina is the presence of putative NK1 autoreceptors expressed by SP-containing amacrine cells.     

Glucagon-like immunoreactivity in mouse and rat retina

Mouse and rat retinae were examined by the peroxidase-anti-peroxidase technique of immunocytochemistry using an antiserum against glucagon. The immunoreactivity was found in the cells of the ganglion cell layer and inner nuclear layer, including Müller cells. These observations may indicate that glucagon or a similar peptide is important in neuromodulation and/or metabolism of retinal cells.     

Cell type-specific effects of Rb deletion in the murine retina

Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. Here we studied the role of Rb in normal retinal development and in retinoblastoma using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb led to more widespread retinal apoptosis, and adults showed loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina did not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina caused retinal dysplasia or retinoblastoma.     

The distribution of c-myb immunoreactivities in the adult mouse retina

The myb gene family is composed of three different myb-related genes, A-, B- and c-myb. Among these, the presence of c-myb mRNA in developing and adult retina was previously reported. However, further study on the expression of c-myb in the retina is warranted because the previous study was only performed on developing retinal tissues by in situ hybridization technique. Therefore, in this study, we tried to perform immunohistochemical study, with a focus on the c-myb protein expression in adult retina. Although the cell types were unconfirmed, c-myb protein was likely to have been found in the ganglion, amacrine, horizontal and photoreceptor cells, judging from their locations and morphologies. The experimental data suggested that c-myb immunoreactivities were expressed by the cells in the neural retina, even in the adult stage, especially within some types of cells in specified retinal layers. This suggests that c-myb might play a role in the physiology of the retinal cells, not only in differentiation during retinal development.     

Folate antagonist,methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.     

Mapping differentiation kinetics in the mouse retina reveals an extensive period of cell cycle protein expression in post‐mitotic newborn neurons

Background: Knowledge of gene expression kinetics around neuronal cell birth is required to dissect mechanisms underlying progenitor fate. Here, we timed cell cycle and neuronal protein silencing/induction during cell birth in the developing murine retina. Results: The pan‐cell cycle markers Pcna and Mcm6 were present in the post‐mitotic ganglion cell layer. Although confined to the neuroblastic layer (NBL), 6–7% of Ki67⁺ cells lacked six progenitor/cell cycle markers, and expressed neuronal markers. To define protein extinction/induction timing, we defined G2/M length throughout retinogenesis, which was typically 1–2 h, but <10% cells took double this time. BrdU‐chase analyses revealed that at E12.5, Tubb3 (Tuj1) appeared at M‐phase, followed by Calb2 and Dcx at ～2 h, Elavl2/3/4 at ～4 h, and Map2 at ～6 h after cell birth, and these times extended with embryonic age. Strikingly, Ki67 was not extinguished until up to a day after cell cycle exit, coinciding with exit from the NBL and induction of late markers such as Map1b/Uchl1/Rbfox3. Conclusions: A minor population of progenitors transits slowly through G2/M and, most importantly, some cell cycle proteins are retained for an unexpectedly long period in post‐mitotic neurons. The high‐resolution map of cell birth kinetics reported here provides a framework to better define mechanisms that regulate neurogenesis. Developmental Dynamics 241:1525–1544, 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of transgenic mouse lines expressing Cre recombinase in the retina

The mammalian retina consists of five major classes of neuronal cells, as well as glial cells, and it contains more than 50 cell types. The ability to manipulate gene expression in specific cell type(s) in the retina is important for understanding the molecular mechanisms of retinal function and diseases. The Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, over-expression, and ectopic expression in vivo in a cell- and tissue-specific fashion. The key to this tool is the availability of Cre mouse lines with cell- or tissue-type specific expression of Cre recombinase. To date, a large number of Cre-transgenic mouse lines have been generated to target Cre recombinase expression to specific neuronal and glial cell populations in the central nervous system; however, information about the expression patterns of Cre recombinase lines in the retina is largely lacking. In this study, we examined and characterized the expression patterns of Cre recombinase in the retinas of 15 Cre-transgenic mouse lines. Significant Cre-induced recombination or expression of Cre recombinase was observed in the majority of these lines. In particular, we found several Cre lines in which the Cre-induced recombination was found to target exclusively or predominantly a single type or class of retinal cells, including bistratified retinal ganglion cells, starburst amacrine cells, rod bipolar cells, and Müller glial cells. In other lines, the Cre-induced recombination was found in several retinal cell types. These Cre lines provide a valuable resource for retinal research.     

Cell differentiation in pancreatic cancer

Cell differentiation in cancer of the exocrine pancreas is currently under intensive study. In this report, new developments are critically reviewed on the use of transplantable pancreatic carcinoma and the hybridoma technique to define cell membrane changes during pancreatic cancer growth. It is concluded that two categories of plasmalemma structure hold promise as differentiation markers specific for pancreatic cancer: (1) membrane receptors for cholinergic and peptide secretagogues and (2) membrane glycoproteins as detected by monoclonal antibodies. Assay of secretagogue receptors and membrane glycoprotein antigens will be central to elucidation of mechanisms of pancreatic carcinoma cell differentiation (? stem cell differentiation or retrodifferentiation) and, hopefully, will provide tumor-specific or tissue-specific markers for the laboratory diagnosis and monitoring of pancreatic cancer.     

Spontaneous oscillatory activity of starburst amacrine cells in the mouse retina

Using patch-clamp techniques, we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of approximately 3.5 Hz and an average maximal amplitude of approximately 120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by approximately 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons.