Toward whole tissue imaging of axolotl regeneration

The axolotl is a highly regenerative organism and has been studied in laboratories for over 150 years. Despite a long-standing fascination with regeneration in general and axolotl specifically, we are still scratching the surface trying to visualize and understand the complex cellular behavior that underlies axolotl regeneration. In this review, we will discuss the progress that has been made in visualizing these processes focusing on four major aspects: cell labeling approaches, the removal of pigmentation, reductionist approaches to perform live cell imaging, and finally recent developments applying tissue clearing strategies to visualize the processes that underly regeneration. We also provide several suggestions that the community could consider exploring, notably the generation of novel alleles that further reduce pigmentation as well as improvements in tissue clearing strategies.     

Gene expression profile of the regeneration epithelium during axolotl limb regeneration

Urodele amphibians are unique among adult vertebrates in their ability to regenerate missing limbs. The process of limb regeneration requires several key tissues including a regeneration‐competent wound epidermis called the regeneration epithelium (RE). We used microarray analysis to profile gene expression of the RE in the axolotl, a Mexican salamander. A list of 125 genes and expressed sequence tags (ESTs) showed a ≥1.5‐fold expression in the RE than in a wound epidermis covering a lateral cuff wound. A subset of the RE ESTs and genes were further characterized for expression level changes over the time‐course of regeneration. This study provides the first large scale identification of specific gene expression in the RE. Developmental Dynamics 240:1826–1840, 2011. © 2011 Wiley‐Liss, Inc.     

多疣壁虎断尾再生中巨噬细胞的组织定位

目的:研究巨噬细胞与多疣壁虎断尾再生的关系.方法:多疣壁虎120只,断尾后随机分为野生型组(正常组)、生理盐水组(免疫正常组)、生理盐水+氢化可的松组(免疫抑制组)和生理盐水+脂多糖组(免疫增强组).免疫抑制组腹腔注射氢化可的松80μg·g-1·d-1;免疫增强组注射脂多糖10 μg·g-1·d-1;采用免疫组织化学检测巨噬细胞组织定位.结果:在壁虎断尾再生过程中,横断创面巨噬细胞的浸润定位层次是改变的,断尾12 h后迁移到尾巴断截面附近的巨噬细胞数量达到峰值.免疫正常组、增强组在断尾后24 h与免疫抑制组相比较,断面附近巨噬细胞的数量显著增加.结论:巨噬细胞有可能参与多疣壁虎断尾再生过程.     

Spatiotemporal regulation of keratin 5 and 17 in the axolotl limb

Background : Amphibians have greater regeneration capability than higher vertebrates. They can regenerate their limbs after an amputation. As a limb is regenerated, a regeneration‐specific epithelium called the apical epithelial cap (AEC) is induced. The AEC is an essential structure for limb regeneration. Despite the importance of the AEC, molecular marker genes have not been well studied at the molecular level. Results : In the present study, keratin5 (KRT5) and KRT17 were investigated in an axolotl‐regenerating limb. KRT5 and KRT17 were expressed in a regenerating limb but down‐regulated in a differentiating limb. KRT5 showed characteristic regulation in a regenerating blastema. KRT5 was suppressed in the basal layer of the AEC. This KRT5 suppression was correlated to the blastema differentiation and nerve presence. Simple skin wounding could also upregulate both KRT5 and KRT17 gene expression. But these genes were suppressed within a shorter time than in limb regeneration. Conclusions : The KRT5 and KRT17 gene profile can be a useful marker gene to investigate AEC in limb regeneration. Developmental Dynamics 241:1616–1624, 2012. © 2012 Wiley Periodicals, Inc.     

In vivo monitoring of neural stem cells after transplantation in acute cerebral infarction with dual-modal MR imaging and optical imaging

Stem cell therapies are promising strategies for the treatment of stroke. However, their clinical translation has not been fully realized due, in part, to insufficient ability to track stem cell migration and survival longitudinally over long periods of time in vivo. In this work, we synthesized a new class of manometer-sized cationic polymersomes loaded with superparamagnetic iron oxide nanoparticles and quantum dots for in vivo dual-modal imaging of stem cells. The results demonstrated that the synthesized cationic polymersomes can act as an effective and safety carrier to transfer image labels into neural stem cells, upon which the distribution and migration of grafted stem cells could be monitored by MR imaging up to 6 weeks and by fluorescence imaging within 4 weeks in the context of ischaemic brain injury. Cationic polymersomes hold great promise in the longitudinal monitoring of transplanted stem cells by using dual-modal MRI and optical imaging.     

A comparative study of gland cells implicated in the nerve dependence of salamander limb regeneration

Limb regeneration in salamanders proceeds by formation of the blastema, a mound of proliferating mesenchymal cells surrounded by a wound epithelium. Regeneration by the blastema depends on the presence of regenerating nerves and in earlier work it was shown that axons upregulate the expression of newt anterior gradient (nAG) protein first in Schwann cells of the nerve sheath and second in dermal glands underlying the wound epidermis. The expression of nAG protein after plasmid electroporation was shown to rescue a denervated newt blastema and allow regeneration to the digit stage. We have examined the dermal glands by scanning and transmission electron microscopy combined with immunogold labelling of the nAG protein. It is expressed in secretory granules of ductless glands, which apparently discharge by a holocrine mechanism. No external ducts were observed in the wound epithelium of the newt and axolotl. The larval skin of the axolotl has dermal glands but these are absent under the wound epithelium. The nerve sheath was stained post-amputation in innervated but not denervated blastemas with an antibody to axolotl anterior gradient protein. This antibody reacted with axolotl Leydig cells in the wound epithelium and normal epidermis. Staining was markedly decreased in the wound epithelium after denervation but not in the epidermis. Therefore, in both newt and axolotl the regenerating axons induce nAG protein in the nerve sheath and subsequently the protein is expressed by gland cells, under (newt) or within (axolotl) the wound epithelium, which discharge by a holocrine mechanism. These findings serve to unify the nerve dependence of limb regeneration.     

In vivo real-time visualization of mesenchymal stem cells tropism for cutaneous regeneration using NIR-II fluorescence imaging

Mesenchymal stem cells (MSCs) have shown great potential for cutaneous wound regeneration in clinical practice. However, the in vivo homing behavior of intravenously transplanted MSCs to the wounds is still poorly understood. In this work, fluorescence imaging with Ag₂S quantum dots (QDs) in the second near-infrared (NIR-II) window was performed to visualize the dynamic homing behavior of transplanted human mesenchymal stem cells (hMSCs) to a cutaneous wound in mice. Benefiting from the desirable spatial and temporal resolution of Ag₂S QDs-based NIR-II imaging, for the first time, the migration of hMSCs to the wound was dynamically visualized in vivo. By transplanting a blank collagen scaffold in the wound to help the healing, it was found that hMSCs were slowly recruited at the wound after intravenous injection and were predominantly accumulated around the edge of wound. This resulted in poor healing effects in terms of slow wound closure and thin thickness of the regenerated skin. In contrast, for the wound treated by the collagen scaffold loaded with stromal cell derived factor-1α (SDF-1α), more hMSCs were recruited at the wound within a much shorter time and were homogenously distributed across the whole wound area, which enhances the re-epithelialization, the neovascularization, and accelerates the wound healing.     

Reorganization of the ependyma during axolotl spinal cord regeneration: changes in intermediate filament and fibronectin expression.

Changes in intermediate filament content and extracellular matrix material showed that the injury response of ependymal cells in lesioned axolotl spinal cord involves an epithelial-to-mesenchymal transformation, and that fibrous astrocytes are excluded from the remodeling lesion site. Antibody localization was used to visualize cytokeratin-, vimentin-, and glial fibrillary acidic protein- (GFAP-) containing intermediate filaments, as well as the adhesive glycoprotein fibronectin. In normal axolotl spinal cord cytokeratins were found near the apical surface of the ependymal cells. Transmission electron microscopic examination suggested that these cytokeratins were in tonofilaments. Cytokeratin expression was lost and vimentin production was initiated in ependymal cells 2-3 weeks following spinal cord injury. There was a period of approximately 1-2 weeks when cytokeratins and vimentin were co-expressed in vivo. This co-expression was maintained in vitro by culture on a fibronectin-coated substratum. As the central canal reformed, vimentin expression was lost. Ependymal cells lacked GFAP intermediate filaments, but GFAP was present in fibrous astrocytes of the neuropil and white matter. Following injury, GFAP localization showed that fibrous astrocytes disappeared from the remodeling lesion site and reappeared only after the ependymal epithelium reformed and newly myelinated axons were found. Fibronectin expression closely followed the expression of vimentin during mesenchymal ependymal cell outgrowth. These results suggest that the ependymal cell outgrowth requires changes in cell shape followed by changes in production of extracellular matrix.     

Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up‐regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan‐Trk inhibitor K252a or the TrkA inhibitor GW‐441756, but not by the TrkB inhibitor ANA‐12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.     

BMP-4 and Noggin signaling modulate dorsal fin and somite development in the axolotl trunk.

BMP-4, a member of the TGF-beta superfamily of growth factors, is involved in various developmental processes. We investigated the effects of BMP-4 and its antagonist Noggin on axolotl trunk development. Implantation of BMP-4-coated microbeads caused inhibition of muscle and dorsal fin formation in the vicinity of the microbeads. At some distance, myotomes developed with reduced height but increased width, which was accompanied by increased cell proliferation. These effects could be modulated by co-implanting Noggin-coated beads. Immunostaining of Pax7 further revealed that although the dermomyotome was absent in the vicinity of BMP-4-coated beads, at some distance from them, it was thicker than in controls, indicating that moderate amounts of BMP-4 stimulate this layer of undifferentiated cells. In contrast, Noggin generally inhibited the dermomyotome, possibly indicating premature differentiation of dermomyotome cells. We conclude that BMP-4 and Noggin are involved in the regulation of cell proliferation and differentiation during somite development.     

Elastoidin turn-over during tail fin regeneration in teleosts

Summary During teleostean fin regeneration the actinotrichia, rods of a collagen-like protein, the elastoidin, are immersed in the blastema, maintaining their apical position. In this epimorphic event the latter fact might be achieved by either a cellular carriage or a continuous turn-over of these hyperpolimerized fibrils. By means of a ³H-proline pulse and radioautographic chase experiment of the isolated actinotrichia we have found a turn-over of collagen within the structure. From these and additional morphometric results, we present in this work an operational hypothesis of how gradually differentiating blastemal cells and an appropriately shaped basal lamina, can control the number and distribution of actinotrichia which might be under the balanced control of their synthesis and degradation.     

细胞标记与体内追踪成像：在动物和人类中应用的最新进展

文题释义：细胞治疗：采用生物工程方法获取细胞,再通过体外扩增、特殊培养等处理后,使这些细胞具有增强免疫、杀死病原体和肿瘤细胞、促进组织器官再生和机体康复等治疗功效,从而达到治疗疾病的目的。 分子影像学：运用影像学对活体状态下的生物过程进行细胞和分子水平的定性和定量研究。以药物分子作为探针进行成像,能够得到药物的药代动力学、生物分布、靶向性、毒性等众多信息,从而帮助缩短药物研发周期、降低失败风险。 背景：随着国内外多个细胞治疗产品获得临床试验批准,细胞治疗例如干细胞治疗、肿瘤过继免疫疗法得到广泛关注。体内细胞实时观察与成像可以直观显示细胞的分布,追踪细胞的活动,监测细胞的活性,观察细胞的迁移和生长。目前的许多成像技术可以使体内细胞可视化,例如超声、光学、磁共振成像以及核成像技术,这些方法均需对应不同的标记和检测策略,每种策略都有其固有的优点和缺点。 目的：结合最新的研究动态,将对不同细胞追踪方法的原理、发展和这些方法在动物和人类中应用的最新进展进行综述。 方法：第一作者以“cell tracking,in vivo cell tracking,PET imaging,MRI,optical imaging”为关键词在PubMed、Google Scholar、Web of Science、中国知网等数据库进行检索,重点关注过去5-10年的相关文章。文章内容主要描述不同细胞追踪方法的原理以及在动物模型和临床患者体内的细胞追踪应用。 结果与结论：在过去的二十几年中,细胞追踪已经发展成为一个多方面的学科,不仅在动物模型中建立了多种稳健的方法,并且在人类的一些研究中证明了临床转化的可行性。尤其是以PET、MRI成像技术为代表的无创检测,新型对比剂的研发,为细胞治疗在临床与科研的应用提供了强有力的支持。 ORCID: 0000-0002-3764-0154(徐梦欣) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程