Conditioned medium-mediated photoreceptor differentiation in retina from embryonic rd chickens

Retina cells from 8-day rd (retinal degenerate) chicken embryos were cultured in media supplemented with optic lobe conditioned medium (OLCM). The morphogenesis of rd photoreceptors is being described. Several differentiating photoreceptors depicted a membranous sac protruding from the apical end of the cell as revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Such structures were found mostly in 8-day-old cultures supplemented with OLCM and were absent in controls. They resembled rudimentary outer segments emanating from a cilium at the apical inner segments. TEM showed a few stacks of free floating disks within the membranous structure which was suggestive of a rudimentary outer segment development. The possible neurotrophic effect of OLCM on rd retina photoreceptor differentiation is being suggested.     

Muscarinic acetylcholine responses in the early embryonic chic retina

The action of acetylcholine on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in early embryonic chick retinae. Whole neural retinae were isolated from embryonic day 3 (E3) chicks and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). Increases in [Ca²⁺]_i were evoked by the puff application of acetylcholine at concentration than 0.1 μM. The Ca²⁺ response became larger in dose–dependant manner up to 10 μM of acetylcholine applied. The rise in [Ca²⁺]_i was not due to the influx of Ca⁺² through calcium channels, but to the release of Ca²⁺ from internal stores. A calcium channel antagonist, nifedipine, which completely blocks the Ca²⁺ rise caused by depolarization with 100 mM K⁺, had no effects on the acetylcholine response and the Ca²⁺ response to acetylcholine occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolished the response to 10 μM acetylcholine, whereas d-tubocurarine of 100 μM had no effects. Two muscarinic agonists, muscarine and carbamylcholine (100 μM each), evoked comparable responses with that to 10 μM acetylcholine. The developmental change of the muscarinic response was examined from E3 to E13. The Ca2+ response to 100 μM carbamylcholine was intense at E3-E5, then rapidly declined until E8. The muscarinic Ca²⁺ mobilization we found in the early embryonic chick retina may be regarded as a part of the “embryonic muscarinic system” proposed by Drew's group, which appears transiently and ubiquitously at early embryonic stages in relation to organogenesis. 1994 John Wiley & Sons, Inc.     

胚胎干细胞向心肌细胞分化过程中能量代谢变化的研究

本文通过定向诱导人胚胎干细胞分化为心肌细胞,对分化过程中胚胎干细胞、心肌祖细胞和心肌细胞糖酵解能力和线粒体氧化磷酸化能力进行实时定量检测,旨在探索分化过程中细胞能量代谢表型的转换机制.用GSK3抑制剂CHIR99021和Wnt信号通路小分子抑制剂IWP2的方法定向分化人胚胎干细胞为心肌祖细胞和心肌细胞;细胞免疫荧光检测人胚胎干细胞标志物,流式细胞术检测人心肌祖细胞和心肌细胞标志物;应用细胞外流量分析(extracellular flux analysis)方法检测人胚胎干细胞、心肌祖细胞和心肌细胞能量代谢情况.研究发现,人胚胎干细胞干性保持稳定,均表达Nanog、OCT4、SOX2细胞标志物;在向心肌分化过程中,第7 d心肌祖细胞标志物Isl1表达99%以上,分化第14 d心肌细胞标志物cTnT表达83%以上;人胚胎干细胞糖酵解代谢能力最强,心肌细胞线粒体功能最强,心肌祖细胞处于两种代谢方式的过度阶段.因此推断,在人胚胎干细胞向心肌细胞分化的过程中,细胞糖酵解能力逐渐减弱,线粒体氧化磷酸化能力逐渐增强,细胞的能量代谢类型发生转变.本研究旨在优化人胚胎干细胞定向分化为心肌细胞的方法,揭示...     

Metabolic requirements for differentiation of embryonic sympathetic ganglia cultured in the absence of exogenous nerve growth factor.

The embryonic mouse superior cervical ganglion (SCG) in culture was employed to define the role of ongoing metabolic processes in morphological and biochemical development. The 14 gestational day SCG does not require added nerve growth factor (NGF) for differentiation in vitro. Consequently, its use allows study of intraganglionic regulation of neuronal growth in the absence of complicating, exogenous growth factors. Ganglia were cultured without added NGF, in medium containing various metabolic inhibitors; neurite elaboration and development of tyrosine hydroxylase (T-OH) activity, a biochemical marker of adrenergic maturation, were evaluated. Neurite elaboration proceeded normally with inhibition of RNA synthesis by actinomycin D, or of protein synthesis by cycloheximide or puromycin. In contrast, inhibition of RNA or protein synthesis prevented normal development of T-OH activity. However, neurites and T-OH developed normally in the presence of DNA synthesis inhibition by cytosine arabinoside, which markedly reduced the nonneuronal cell population. These observations suggest that neurite elaboration and the ontogenetic increase in T-OH activity are regulated differently in ganglia cultured in the absence of exogenous NGF. Moreover, the initial outgrowth of neurites and increase in T-OH activity may occur independent of peripherally migrating support cells in this embryonic ganglion.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

从胚胎干细胞到视网膜

视网膜退行性病变影响着全世界数百万人。然而,视网膜是人体再生能力很差的一类组织,成年机体无法自我更新那些病变中丢失的视网膜细胞,导致视网膜退行性病变的不可逆性。因此,恢复患者视觉将依赖于引入外源细胞替代丢失的视网膜神经元。胚胎干细胞（ES细胞）具有无限的自我更新能力和形成机体所有类型细胞的巨大分化潜力。这两个特性使得ES细胞成为细胞替代疗法的理想供体细胞。近年来,人们在探索将ES和诱导多能干细胞（iPS细胞）体外定向诱导分化为视网膜神经元,甚至整个视网膜方面已取得多项进展,并且体外形成的视网膜细胞可以与宿主视网膜整合。在此篇综述中,首先简要概括哺乳动物视网膜的组织结构、发育过程和调控机制,然后,重点阐述近年来科研工作者探索ES/iPS细胞体外诱导分化为视网膜细胞和组织的研究进展。     

Ligatin from embryonic chick neural retina

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.     

Histogenesis and dependent glutamine synthetase inducibility in embryonic neural retina. Irreversible inhibition of differentiation by 5-bromodeoxyuridine

Young, mitotically active neural retinas from 7-day chick embryos were cultured with 5-bromodeoxyuridine (BrdU) for 8 hr or more. After this treatment, they failed to differentiate beyond the stage at which they were explanted; there was no histogenesis or increase in glutamine synthetase (GS) inducibility in intact tissues or in aggregates of dissociated cells. Normally GS can be induced in the retina with hydrocortisone as the cells cease to be mitotically active and begin showing histological organization after day 7. This inhibition by BrdU was irreversible even in the presence of excess thymidine. Overall incorporation of ¹⁴C-amino acids into protein was only slightly inhibited, and the ability of cells from treated tissue to aggregate and sort out from nonneural cell types was unaffected. Control cultures without BrdU showed considerable histogenesis and a parallel increase in enzyme inducibility. Postmitotic 10-day retinas appeared to be unaffected by BrdU. The incorporation rates of both tritiated BrdU and thymidine (dT) into DNA were 14× higher in 7- than in 10-day retinas. Simultaneous addition of excess unlabeled dT with either of the labeled nucleosides reduced their incorporation and reduced the inhibitory action of BrdU on differentiation.It is concluded that BrdU irreversibly inhibits the differentiation of retina cell surface properties involved in histogenesis and dependent cytodifferentiation without affecting already differentiated properties of the cell surface. The results support the hypothesis that histogenesis is directed by genes affecting specific cell surface properties.     

NCAM in the differentiation of embryonic lens tissue

The role of the neural cell adhesion molecule (NCAM)2 in ocular lens differentiation was investigated in chicken embryos. Changes in expression of NCAM were documented by immunohistology of frozen sections. This analysis revealed that NCAM diminished during lens fiber differentiation, in contrast to the gap junction-associated protein MP26 which became more abundant. The form of NCAM expressed was determined by Western blot analysis of proteins extracted from the different regions of the Embryonic Day 6 lenses. All regions expressed NCAM with an apparent molecular weight of 140 kDa and relatively low levels of polysialylation. The function of NCAM in lens differentiation was investigated using antibodies that inhibit NCAM-mediated adhesion. Two parameters that change during maturation of the lens epithelial cells were monitored: the thickness of the tissue, indicating the length of lens cells, and the particle arrangement of gap junctions, reflecting the state of junctional differentiation. When epithelial cell explants of Embryonic Day 6 lenses were cultured for 5 days, the cells elongated and displayed an increase in the loose, random intramembranous particle arrangements characteristic of maturing lens fiber gap junctions. When the explants were cultured in the presence of anti-NCAM Fabs, the epithelia were thinner than in matched controls and had particle arrangements characteristic of a less mature state. The expression of NCAM during lens differentiation and the effects of attenuating NCAM function suggest that adhesion mediated by NCAM is an essential event in lens cell differentiation.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.     

Oriented extracellular channels and axonal guidance in the embryonic chick retina

Transmission electron microscopy and serial reconstruction of lum sections were used to determine whether aligned extracellular channels precede the outgrowth of optic fibers in the embryonic chick retina. At stage 16, just prior to the migration of optic axons toward the optic stalk, extracellular spaces bounded by neuroepithelial cell processes, in the superficial (vitread) region of the retina, were aligned toward the optic stalk. The optic axons subsequently entered and grew within these spaces. After formation of the ganglion cell fiber layer (GCFL), the growth cones of new optic axons entered the most vitread portion of that layer. Hypertonic fixatives caused shrinkage of cell processes, resulting in intercellular separation. However, growth cone filopodia retained close contacts with neighboring glial cell endfeet and with optic axons in these solutions. This suggests that growth cones may be adherent to these structures. Transmission electron microscopy in conjunction with the use of hypertonic solutions may become a useful technique for assaying intercellular adhesivity.     

Metabolic differentiation in actively swarming Salmonella

Most current paradigms of microbial metabolism have been derived from studying cells grown under a variety of nutrient compositions in aqueous environments. With recent advances in genomics and experimental techniques, alternative forms of bacterial growth are increasingly being explored. When propagated on nutrient-rich semi-solid media, several species of bacteria undergo a morphological differentiation into swarmers that are capable of migrating on surfaces. Recent studies indicate that swarmer differentiation represents much more than a motility phenotype, as several clinically important attributes are also co-regulated. We demonstrate that migrating swarmer cells of Salmonella are metabolically differentiated compared to the vegetative swimmer cells grown in the same nutrient environment. Furthermore, once the cells have differentiated, the swarmers remain in this physiological state under conditions that do not promote the initial differentiation. The bacterium's capacity to override some of the classic paradigms of metabolic regulation established in aqueous environments represents a unique physiological response by the pathogen that may be advantageous in polymicrobial environments such as the host.