Multipotent and Stem Cells in the Developing, Definitive, and Regenerating Vertebrate Eye

This is a review of the experimental studies on the vertebrate retina neurogenesis. Data are provided on the distribution and localization of multipotent and stem cells in the developing, definitive, and regenerating eye. At the early stages of retina development, the neuroepithelial cells divide synchronously, thus leading to the accumulation of a certain number of the retinal rudiment cells. Synchronous divisions precede the asynchronous ones, when the differentiation of the retinal cells is initiated. The neuroepithelial cells are multipotent: the neuroblast is a source of the cells of different types, for example, neurons and glial cells. The proliferating multipotent cells are preserved in the ciliary-terminal zone of the retina of amphibians, fish, and chickens during their entire life. The differentiated pigment epithelium cells also proliferate in this area of the eye. The multipotent cells of the retinal ciliary-terminal zone and cells of the pigment epithelium in the eye periphery provide for the growth of amphibian and fish eyes during the entire life of these animals. In adult mammals, clonable and self-renewable cells were found among the pigmented differentiated cells in the ciliary folds. In a culture, the stem cells form spheroids consisting of depigmented and proliferating cells. Upon transdifferentiation, the cells of spheroids form rods, bipolar cells, and ganglion and glial cells, thus suggesting the possible regenerative potencies of the stem cells in the ciliary body of the mammalian eye. The main event of retinal regeneration in newts is the transdifferentiation of the pigment epithelium cells. The results of comparative analysis suggest that the stem cells of the ciliary body in the mammalian eye and pigment epithelium cells in lower vertebrates exhibit similar potencies and use similar mechanisms during the formation of the cells of the neural series.     

Transdifferentiation Potential of Ciliary and Pigment Epithelial Cells in Lower Vertebrates and Mammals

Cellular composition of the peripheral region of the eye in amphibians and mammals as well as embryonic fissure in amphibians was studied. Different distributions of proliferating cells in retinal pigment epithelium have been revealed in adult amphibians (newt, axolotl, and Xenopus). Single cells incorporated [³H]thymidine in the newt and Xenopus; 0.4% cells, in the axolotl. An embryonic fissure was observed in the eye of the axolotl. Pigment epithelial cells in the embryonic palpebral region actively proliferated: about 20% cells incorporated [³H]thymidine. Proliferating cells were also localized in the ciliary marginal zone of the retina in all studied amphibians, particularly, in the axolotl. In newborn hamsters, [³H]thymidine-labeled cells have been revealed in the pigment epithelium as well as in the outer pigmented and inner unpigmented layers of the ciliary body. Proliferative activity of the peripheral regions of the eye is due to eye growth in adult amphibians and newborn hamsters. After retinectomy, the retina is regenerated from the cells of the growth ciliary marginal zone in all amphibians, pigment epithelial cells in the newt, and pigment epithelial cells of the embryonic fissure in the axolotl. Heterogeneous composition of the pigment epithelium in the newt and axolotl reflects high transdifferentiation potential of these regions. Structural comparison of the peripheral region of the eye in amphibians and mammals demonstrate that the ciliary body of mammals containing stem cells is homologous to the ciliary marginal zone of amphibians containing multipotent cells.     

Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation

Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/β-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.     

Protection from lipopolysaccharide-induced pulmonary microvascular endothelial cell injury by activation of hedgehog signaling pathway

Pulmonary microvascular endothelial cells (PMVECs) are critically involved in the pathogenesis of acute lung injury. Hedgehog signaling pathway plays a fundamental role in embryonic development as well as adult morphogenesis and carcinogenesis. As the priming protein of hedgehog signaling pathway, sonic hedgehog (Shh) may recently be advantage for decreasing endothelial injury and promoting the repair of endothelial barrier function. To investigate the expression and role of hedgehog signal pathway in PMVECs injured by lipopolysaccharide (LPS), cells were divided into six groups: control group, LPS group, rhShh group, LPS + rhShh group, rhShh + cyclopamine group, and LPS + rhShh + cyclopamine group. Real time RT-PCR and Western blotting were used to detect the mRNA and protein expression of hedgehog signal molecules including Shh, Patched-1 (Ptc-1) and Gli1 in nucleus. The activity of PMVECs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In this study, we found that Shh, Ptch1, and Gli1 were expressed in rat PMVECs and their expression decreased when cells were treated by LPS. In the other hand, LPS inhibited the activity of rat PMVECs and caused the cells injury. Activation of Hedgehog signaling pathway by Shh could elevate the activity of PMVECs with pretreatment by LPS. Therefore, hedgehog signaling pathway should play a protective role on injury PMVECs by LPS.     

抑制Hedgehog信号通路改变结肠癌细胞基因表达谱

Hedgehog（Hh）信号通路在机体发育和肿瘤发生中发挥着重要作用。在该研究中,Western blot检测三株结肠癌细胞Hedgehog信号通路组分的表达,结果表明三株结肠癌细胞中HT-29细胞Hedgehog信号通路组分较完整。采用MTT和BrdU法检测Hedgehog信号通路膜受体Smo特异性抑制剂环杷明和末端转录因子Gli1/2的特异性抑制剂GANT61对HT-29细胞的影响,提示这两种抑制剂均显著抑制HT-29细胞生存率和细胞增殖率,且GANT61比环杷明更敏感。表达谱芯片检测阻断Hedgehog信号通路后HT-29细胞基因谱的变化,结合生物信息学分析,揭示HT-29细胞经环杷明和GANT61处理后基因表达呈现抑制特征,其差异基因表达主要以下调为主,其中环杷明主要影响细胞内源刺激等,而GANT61主要影响代谢和类固醇合成,并与MAPK信号通路有关联,两者均能影响细胞免疫及凋亡相关通路。这些结果提示,Hh信号通路有可能作为结肠癌的治疗靶点。     

Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells

Pluripotent mesenchymal cells form a population of precursors to a variety of cell types, including osteoblasts and adipocytes. Aging tilts the balance in favor of adipocyte differentiation at the expense of osteoblast differentiation, resulting in reduced bone formation and osteopenic disorders, including osteoporosis, in humans and animals. Understanding the mechanisms involved in causing this apparent shift in differentiation and identifying factors that stimulate osteoblast formation while inhibiting adipogenesis are of great therapeutic interest. In this study we report that specific, naturally occurring oxysterols, previously shown to direct pluripotent mesenchymal cells toward an osteoblast lineage, exert their osteoinductive effects through activation of Hedgehog signaling pathway. This was demonstrated by 1) oxysterol-induced expression of the Hh target genes Gli-1 and Patched, 2) oxysterol-induced activation of a luciferase reporter driven by a multimerized Gli-responsive element, 3) inhibition of oxysterol effects by the hedgehog pathway inhibitor, cyclopamine, and 4) unresponsiveness of Smoothened-/- mouse embryonic fibroblasts to oxysterols. Using Patched-/- cells that possess high baseline Gli activity, we found that oxysterols did not dramatically shift the IC50 concentration of cyclopamine needed to inhibit Gli activity in these cells. Furthermore, binding studies showed that oxysterols did not compete with fluorescently labeled cyclopamine, BODIPY-cyclopamine, for direct binding to Smoothened. These findings demonstrate that oxysterols stimulate hedgehog pathway activity by indirectly activating the seven-transmembrane pathway component Smoothened. Osteoinductive oxysterols are, therefore, novel activators of the hedgehog pathway in pluripotent mesenchymal cells, and they may be important modulators of this critical signaling pathway that regulates numerous developmental and post-developmental processes.     

Repression of Hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation

Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2δC2), increased T-cell differentiation and activation in response to TCR signalling. Expression of the Gli2δC2 transgene increased differentiation from CD4+CD8+ to single positive thymocyte, and increased peripheral T cell populations. Gli2δC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate.     

Patterns of cytokeratin and vimentin expression in the human eye

We studied the expression of the various cytokeratin (CK) polypeptides and vimentin in tissues of the human eye by applying immunocytochemical procedures using a panel of monoclonal antibodies as well as by performing biochemical analyses of microdissected tissues. Adult corneal epithelium was found to contain significant amounts of the cornea-specific CKs nos. 3 and 12 as well as CK no. 5, and several additional minor CK components. Among these last CKs, no. 19 was found to exhibit an irregular mosaic-like staining pattern in the peripheral zone of the corneal epithelium, while having a predominantly basal distribution in the limbal epithelium. Both the fetal corneal epithelium and the conjunctival epithelium were uniformly positive for CK no. 19. In the ciliary epithelium, co-expression of CKs nos. 8 and 18 and vimentin was detected, whereas in the retinal pigment epithelium, CKs nos. 8 and 18 were dominant. The present data illustrate the remarkable diversity and complexity of CK-polypeptide expression in the human eye, whose significance with respect to histogenetic and functional aspects is, as yet, only partially clear. The unusual distribution of CK no. 19 in different zones of the corneal epithelium may be related to the specific topography of corneal stem cells. The occurrence of the expression of simple-epithelium CKs in the ciliary and pigment epithelium demonstrates that, despite their neuroectodermal derivation, these are true epithelia.     

Hedgehog参与西伯利亚鲟侧线机械和电感受器分化的初始证据

为揭示Hedgehog（Hh）信号与神经丘和壶腹器官分化的关系,研究以西伯利亚鲟（Acipenser baerii Brandt）为模型,首先对再生过程中的神经丘和壶腹器官的转录组进行比较分析,发现Hh信号通路关键基因（Shh、Patched 1）在两类感受器中差异表达,且它们的表达在再生过程中呈现动态性。然后用环巴胺（Cyclopamine,Hh信号抑制剂）处理西伯利亚鲟胚胎（st29）,用扫描电镜和FM1-43荧光染色对西伯利亚鲟仔鱼（st43-st44）分析发现环巴胺显著抑制了壶腹器官的发育。整体原位杂交表明,Shh、Patched1、Smoothened、Gli2在腹面侧线区域的表达受到了环巴胺的抑制。以上结果暗示Hh信号通路与神经丘和壶腹器官的发育有关,推测Hh信号在神经丘和壶腹器官的分化过程中起到了重要作用。     

Patterns of cytokeratin and vimentin expression in the human eye

Summary We studied the expression of the various cytokeralin (CK) polypeptides and vimentin in tissues of the human eye by applying immunocytochemical procedures using a panel of monoclonal antibodies as well as by performing biochemical analyses of microdissected tissues. Adult corneal epithelium was found to contain significant amounts of the cornea-specific CKs nos. 3 and 12 as well as CK no. 5, and several additional minor CK components. Among these last CKs, no. 19 was found to exhibit an irregular mosaiclike staining pattern in the peripheral zone of the corneal epithelium, while having a predominantly basal distribution in the limbal epithelium. Both the fetal corneal epithelium and the conjunctival epithelium were uniformly positive for CK no. 19. In the ciliary epithelium, co-expression of CKs nos. 8 and 18 and vimentin was detected, whereas in the retinal pigment epithelium, CKs nos. 8 and 18 were dominant. The present data illustrate the remarkable diversity and complexity of CK-polypeptide expression in the human eye, whose significance with respect to histogenetic and functional aspects is, as yet, only partially clear. The unusual distribution of CK no. 19 in different zones of the corneal epithelium may be related to the specific topography of corneal stem cells. The occurrence of the expression of simple-epithelium CKs in the ciliary and pigment epithelium demonstrates that, despite their neuroectodermal derivation, these are true epithelia.Supported by a grant from the Deutsche Forschungsgemeinschaft (Mo 345-3).     

A novel antigen is shared by retinal pigment epithelium and pigmented neural crest

 Pigment cells in vertebrate embryos are formed in both the central and peripheral nervous system. The neural crest, a largely pluripotent population of precursor cells derived from the embryonic neural tube, gives rise to pigment cells which migrate widely in head and trunk.The retinal pigment epithelium is derived from the optic cup, which arises from ectoderm of the neural tube. We have generated an antibody, ips6, which stains an antigen common to pigment cells of retinal pigment epithelium and neural crest. Ips6 stains retinal pigment epithelium and choroid as well as a subset of crest cells that migrate in pathways typical of melanoblasts. Immunoreactivity is seen first in the eye and later in a subset of migrating crest cells. Crest cells in the amphibian embryo migrate along specific, stereotyped routes; ips6 immunoreactive cells are found in some but not all of these pathways. In older wild-type embryos, cells expressing ips6 appear coincident with pigment-containing cells in the flank, head, eye and embryonic gut. In older animals, staining in the eye extends to the intraretinal segment of optic nerve and interstices between photoreceptors and cells at the retinal periphery. We suggest that the ips6 antibody defines an antigen common to pigment cells of central and peripheral origin. Received: 22 January 1996/Accepted: 15 July 1996     

Zebrafish blowout provides genetic evidence for Patched1-mediated negative regulation of Hedgehog signaling within the proximal optic vesicle of the vertebrate eye

In this study, we have characterized the ocular defects in the recessive zebrafish mutant blowout that presents with a variably penetrant coloboma phenotype. blowout mutants develop unilateral or bilateral colobomas and as a result, the retina and retinal pigmented epithelium are not contained within the optic cup. Colobomas result from defects in optic stalk morphogenesis whereby the optic stalk extends into the retina and impedes the lateral edges of the choroid fissure from meeting and fusing. The expression domain of the proximal optic vesicle marker pax2a is expanded in blowout at the expense of the distal optic vesicle marker pax6, suggesting that the initial patterning of the optic vesicle into proximal and distal territories is disrupted in blowout. Later aspects of distal optic cup formation (i.e. retina development) are normal in blowout mutants, however. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway, as the underlying cause of the blowout phenotype. Expanded domains of expression of the Hedgehog target genes patched1 and patched2 were observed in blowout, consistent with a loss of Patched1 function and upregulation of Hedgehog pathway activity. Moreover, colobomas in blowout could be suppressed by pharmacologically inhibiting the Hedgehog pathway with cyclopamine, and maximal rescue occurred when embryos were exposed to cyclopamine between 5.5 and 13 hours post-fertilization. These observations highlight the critical role that Hedgehog pathway activity plays in mediating patterning of the proximal/distal axis of the optic vesicle during the early phases of eye development and they provide genetic confirmation for the integral role that patched1-mediated negative regulation of Hedgehog signaling plays during vertebrate eye development.     

The hedgehog pathway is a modulator of retina regeneration

The embryonic chick has the ability to regenerate its retina after it has been completely removed. Here, we provide a detailed characterization of retina regeneration in the embryonic chick at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). We show that regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2, but we show that it can be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, we show that the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE), as ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, our results suggest that the hedgehog pathway is an important modulator of retina regeneration.