金鱼再生的视神经在顶盖投射精确化的DiI顺行标记研究

本文用微量显微注射法,在金鱼视网膜的背侧用亲脂类荧光染料DiI标记少量神经节细胞,通过顺行标记研究了视神经再生过程中视网膜顶盖投射的精确化过程。在损伤视神经后的不同时期观察了再生视神经纤维在顶盖整装片上的分布。在再生早期它们以超出正常的途径由背腹两侧进入顶盖,广泛分布。但其中大部分仍分布于顶盖腹侧的靶区。在再生晚期通过精确化,重建如正常鱼一样精确的视网膜顶盖投射。这个精确化过程表现在以下三方面:(1)再生于顶盖错误区域的再生视神经纤维的消失;(2)再生早期视神经纤维主干上生长的侧部分支的消失;(3)到达靶区的再生视神经纤维形成重迭的终末分支。由以上结果推测,顶盖中可能存在两类不同的因子:一类是普通诱向因子,存在于整个顶盖中,它在再生早期引导再生的视神经纤维长入顶盖。另一类是神经营养因子,它具区域特异性,在再生晚期引导视神经纤维到达顶盖靶区,形成精确的视网膜顶盖投射。     

Regeneration of the optic tectum in larval stages of Bufo regularis Reuss after partial and total excision

The right optic tectum of four larval stages of Bufo regularis was subjected to partial and total excision to determine the regenerative capacity of the optic tectum. Intense regenerative capacity can be observed at stage 50; the regenerated part grows to a considerable size and its structure is comparable to that of the intact part. This regenerative capacity gradually diminishes and from stage 55 onwards the decrease is pronounced. At stage 57 regenerative capacity is severly reduced and the regenerated part, if present, always has an anomalous structure.     

Morphogenesis and organogenesis in the regenerating planktotrophic larvae of asteroids and echinoids

In a previous study, we described complete body regeneration (with organogenesis) following surgical bisection in the planktotrophic larvae of the asteroids Luidia foliolata and Pisaster ochraceus. Here we present further detailed observations of these unique regenerative processes not presented in the previous paper. Furthermore, we describe for the first time complete regeneration following surgical bisection of planktotrophic larvae of the regular echinoid Lytechinus variegatus and the irregular echinoid Dendraster excentricus. Larvae of both asteroids and echinoids displayed a capacity for rapid regeneration regardless of their developmental stage. Within 48 h after bisection, aggregations of mesenchyme cells with pseudopodia were observed at the site of surgical bisection. These cellular aggregations were similar in appearance to the mesenchymal blastemas that form in adult echinoderms prior to their arm regeneration, and to those described in other deuterostomes that undergo regeneration. When asteroid larvae were surgically bisected in the early stages of their development, clusters of mesenchyme cells developed into completely new pairs of coelomic pouches located anterior to the newly regenerated digestive tract. This indicates that cell fate in regenerating asteroid larvae remains indeterminate during early development. In the larvae of P. ochraceus, regardless of the developmental stage at the time of bisection, both the anterior and posterior portions regenerated all their missing organs and tissues. However, the larvae of L. foliolata displayed differential regenerative capacity in bisected larval halves at the late bipinnaria stage. The differences observed may be due to differences in larval development (L. foliolata has no brachiolaria stage), and may have evolutionary implications. In the regular echinoid L. variegatus, both larval portions regenerated into morphologically and functionally normal larvae that were indistinguishable from non-bisected control larvae. The regenerative processes were similar to those we observed in planktotrophic asteroid larvae. Regenerating larvae readily metamorphosed into normal juveniles. In the irregular echinoid D. excentricus, posterior portions of larvae completed regeneration and metamorphosis, but anterior portions regenerated only partially during the 2-week study. Our observations confirm that asteroid and echinoid larvae provide excellent models for studies of regeneration in deuterostomes.     

Regulation of cardiomyocyte proliferation during development and regeneration

The regulation of cardiomyocyte proliferation is important for heart development and regeneration. The proliferation patterns of cardiomyocytes are closely related to heart morphogenesis, size, and functions. The proliferation levels are high during early embryogenesis; however, mammalian cardiomyocytes exit the cell cycle irreversibly soon after birth. The cell cycle exit inhibits cardiac regeneration in mammals. On the other hand, cardiomyocytes of adult zebrafish and probably newts can proliferate after cardiac injury, and the hearts can be regenerated. Therefore, the ability to reproliferate determines regenerative ability. As in other cells, the relationship between proliferation and differentiation is very interesting, and is closely related to cardiac development, regeneration and homeostasis. In this review, these topics are discussed.     

Optic nerve input‐dependent regulation of neural stem cell proliferation in the optic tectum of adult zebrafish

Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial‐type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU‐positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU‐positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli‐dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Formation of multiple ependymas in the regenerating optic lobe of larvae of the Egyptian toad, Bufo regularis Reuss

In the regenerating optic lobe of Bufo regularis larvae, secondary ependymas were formed in both the dorsal part (optic tectum) and ventral region (tegmentum) of the lobe concerned. These secondary ependymas were frequently observed in the rostral and caudal tectal regions after complete excision of the tectum. Most of the multiple ependymal structures were formed by self-organization of groups of undifferentiated cells migrating from the primary ependyma lining the optic tectum. Others split off from the primary ependyma, but remained in contact with it. The observations emphasize the wide range of possibilities of the cells produced by the larval tectal ependyma in response to partial or total excision of the tectum. The results suggest that cells of ependymal origin, in regenerating tectum, are capable of self-organization to complete ependymal tubes in the absence of direct with the primary ependyma.     

Lgr5+ amacrine cells possess regenerative potential in the retina of adult mice

Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5⁺ cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5⁺ amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Müller glia from Lgr5⁺ amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5⁺ amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age‐related retinal degenerative diseases.     

Proliferative response of the mesencephalic matrix areas in the reparation of the optic tectum of Triturus cristatus carnifex

The localization and proliferative response of optic tectum matrix cells has been studied in adult newt following an experimental lesion on an optic lobe. The results show that 15 days after the lesion the cells in division, autoradiographically labelled, are located in the periventricular layer. Thirty days after the lesion the labelled cells are also found in the innermost grey layers; at 90 days the injured optic tectum regains the cytoarchitecture characteristic of this centre, with labelled cells, whether in the external or in the internal pyriform layers. In all the stages the labelled cells are also found in the periventricular layers of the controlateral optic tectum, in the dorsal pallium and in the striatum. The quantitative data exhibit the existence of a direct relationship between the number of proliferating cells in the injured optic lobe and the extent of the lesion. These data show the possibility of active cellular proliferation for the reconstruction of the lesioned nervous area and for restoration of the characteristic histological structure.     

皮毛之道：以表皮器官为研究模型探讨再生生物学与再生医学的基础概念

再生医学是一个具有巨大潜力的新兴医学领域。该文以此为方向讨论了再生医学研究中的三个关键问题,并以非神经外胚层器官的干细胞行为为例做进一步的探讨。第一,如何获取干细胞,介绍了包括胚胎干细胞、组织干细胞和诱导性多能干细胞的获得途径,以及若干组织细胞重编程的成功范例;第二,如何将干细胞转化为组织和器官,这需要了解干细胞分化以及形态发生的机制,并以羽毛的形态发生为模型,引入了千细胞拓扑生物学的概念以及干细胞微环境调控塑造器官形态的机制;第三,如何将干细胞及其转化产物置于患者体内,并以鼠毛生长周期波为例,阐明了宏观环境因素如何调控干细胞的活性：最后,还分析了在器官发生中干细胞的自组织对于新生毛发组织工程的重要意义。该文的许多原则不仅限于皮肤,同时也适用于其它体内器官。通过对生物再生的过程的基础研究,我们可以受到生物再生之道的启发,逐渐理解组织修复及再生的机制,并提高分子和细胞水平上的干细胞操作技术,希望在不久的将来将干细胞研究成果应用于临床医学。     

New insights into the morphogenic role of stromal cells and their relevance for regenerative medicine. lessons from the heart

The term stromal cells is referred to cells of direct or indirect (hematopoietic) mesenchymal origin, and encompasses different cell populations residing in the connective tissue, which share the ability to produce the macromolecular components of the extracellular matrix and to organize them in the correct spatial assembly. In physiological conditions, stromal cells are provided with the unique ability to shape a proper three‐dimensional scaffold and stimulate the growth and differentiation of parenchymal precursors to give rise to tissues and organs. Thus, stromal cells have an essential function in the regulation of organ morphogenesis and regeneration. In pathological conditions, under the influence of local pro‐inflammatory mediators, stromal cells can be prompted to differentiate into myofibroblasts, which rather express a fibrogenic phenotype required for prompt deposition of reparatory scar tissue. Indeed, scarring may be interpreted as an emergency healing response to injury typical of evolved animals, like mammals, conceivably directed to preserve survival at the expense of function. However, under appropriate conditions, the original ability of stromal cells to orchestrate organ regeneration, which is typical of some lower vertebrates and mammalian embryos, can be resumed. These concepts underline the importance of expanding the knowledge on the biological properties of stromal cells and their role as key regulators of the three‐dimensional architecture of the organs in view of the refinement of the therapeutic protocols of regenerative medicine.     

Neural development in an imaginal disc mutant of Drosophila melanogaster

The neural phenotype of an imaginal disc degenerate mutant l(1)d deg-3 was studied in histological sections. The mutant larvae showed severe abnormalities in the imaginal neural development. Gynandromorphs, which are composed of genetically mutant and nonmutant cells, were generated and analyzed as late larvae. The results of mosaic analysis were consistent with l(1)d deg-3 gene acting autonomously in the imaginal disc and imaginal neural cells. The optic lobe development patterns observed in the larval mosaics provided evidence for an eye disc-optic lobe interaction during the late third instar larval stage.     

Spatio-temporal pattern of programmed cell death in the developing Drosophila optic lobe

A large number of cells die via programmed cell death during the normal development of the Drosophila optic lobe. In this study, we report the precise spatial and temporal pattern of cell death in this organ. Cell death in the developing optic lobe occurs in two distinct phases. The first phase extends from the start of metamorphosis to the mid-pupal stage. During this phase, a large number of cells die in the optic lobe as a whole, with a peak of cell death at an early pupal stage in the lamina and medulla cortices and the region of the T2/T3/C neurons, and a smaller number of dead cells observed in the lobula plate cortex. The second phase extends from the mid-pupal stage to eclosion. Throughout this period, a small number of dying cells can be observed, with a small peak at a late pupal stage. Most of the dying cells are neurons. During the first phase, dying cells are distributed in specific patterns in cortices. The lamina cortex contains two distinct clusters of dying cells; the medulla cortex, four clusters; the lobula plate cortex, one cluster; and the region of the T2/T3/C neurons, one cluster. Many of the clusters maintain their distinct positions in the optic lobe but others extend the region they cover during development. The presence of distinct clusters of dying cells at different phases suggests that distinct mechanisms control cell death during different stages of optic lobe development in Drosophila.     

Optic nerve-dependent changes in adult frog tectal cell phenotypes

Optic nerve activity helps determine the placement of retinal ganglion cell terminals in the optic tectum of the frog. We investigated whether the presence of this nerve might also influence a characteristic of its target structure, neurotransmitter biosynthesis. We performed unilateral optic nerve transections on adult animals and assayed the percent and intensity of substance P- and serotoninlike immunoreactive (SP-ir and 5-HT-ir, respectively) cells in the deafferented and afferented tectal lobes. Regeneration of the optic nerve was prevented. The percent of SP-ir cells in the afferented tectal lobes was significantly less than that in the deafferented ones either 6 weeks or 5 months following optic nerve lesion. Comparison to normal animals indicated that the change in SP-ir expression was due to a decrease in the percent of immunoreactive cells in the afferented tecta ipsilateral to the optic nerve lesion. The serotoninlike immunoreactivity of tectal cells was also significantly different in the two lobes following optic nerve lesions. This difference resulted from an increase in the percent of 5-HT-ir cells in the deafferented tectum. In addition, the intensity of 5-HT-ir cells in the deafferented lobe was significantly greater than in the afferented one. The staining intensity of SP-ir cells underwent only a transient, relative decrease in the deafferented tectum. We conclude that the optic nerve does regulate substance P and serotonin expression in the tectum, but that this regulation likely occurs through different pathways. © 1996 John Wiley & Sons, Inc.     

Morphological and functional characterization of isolated stem cells from the midgut of Chilo suppressalis larvae

The proliferation and differentiation of stem cell populations allow the midgut to grow/regenerate in lepidopteran insect. Basic epithelial regenerative functions can be assessed in vitro by purifying these stem and mature cell populations. Therefore, we isolated and purified stem and mature cells from the midgut of C. suppressalis larvae by density gradient centrifugation and observed the morphologies of these cells. A flow cytometry method was used to monitor C. suppressalis stem cell proliferation and differentiation under different cell culture conditions. We observed high proportions of the stem and differentiating cells in third- and fourth-instar larvae, respectively, indicating that, in larvae, stem cells rapidly proliferate early in development and are strongly differentiated at late stages. Incubation in medium supplemented with fat body extract and ecdysone resulted in a significantly increased proportion of stem cells, not of the differentiating cells, indicating that co-culture with fat body extract and ecdysone stimulates the proliferation of C. suppressalis stem cells. Viability bioassays showed that Cry1Ab displayed significant cytotoxic effects on the midgut cell culture of C. suppressalis. The proportion of differentiating cells was significantly increased after a 48-h exposure to sublethal doses of Cry1Ab toxin, and peaked at the Cry1Ab concentration of 0.3 μg/ml, demonstrating that epithelial cells with strong regenerative capacity via the differentiation of stem cells. These results improve our understanding of C. suppressalis stem cell biology and illustrate the potential role of the enhanced midgut regeneration induced by stem cell proliferation or differentiation as a reparation mechanism to Bt toxin.     

The influence of denervation on grafted hindlimb regeneration of larval Xenopus laevis

The aim of the present research is to ascertain whether in larval Xenopus laevis nerve-independence for the regeneration of early stage limbs and nerve-dependence of late stage limbs observed in a previous work (Filoni and Paglialunga, '90) is related to extrinsic (systemic) factors or to intrinsic changes taking place in the limb cells themselves during development. In this paper the regenerative capacity of early and late stage hindlimbs under the same extrinsic conditions, insofar as both are grafted onto the denervated hindlimbs of host larvae at the same developmental stage, is studied. All the grafted limbs are amputated after the host larvae have reached stage 57-58 (according to Nieuwkoop and Faber, '56). In experiment I, the grafted limb is amputated at stage 52, at the thigh level; in experiment II, the grafted limb is amputated at stage 54-55, at the tarsalia level; in experiment III the grafted limb is amputated at stage 57, at the tarsalia level. In all three experiments, together with the grafted limb, also the host limb is amputated at the tarsalia level. The results show that while grafted limbs amputated at stages 52 and 54-55 regenerate in the absence of nerves, grafted limbs amputated at stage 57 cannot. The failure of late stage grafted limbs to regenerate cannot be explained in terms of an immune-type inhibiting reaction since it has been observed also in denervated autografted limbs and in the host limbs. Since all the grafted limbs are in the same environmental conditions, the results show that in larval Xenopus laevis nerve-independence for regeneration of early stage limbs and nerve-dependence of late stage limbs are not related to factors extrinsic to the limb but to intrinsic changes taking place in the limb cells themselves during development.     

Hair follicle stem cell progeny heal blisters while pausing skin development

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.     

Resting no more: re‐defining telogen,the maintenance stage of the hair growth cycle

The hair follicle (HF) represents a prototypic ectodermal–mesodermal interaction system in which central questions of modern biology can be studied. A unique feature of these stem‐cell‐rich mini‐organs is that they undergo life‐long, cyclic transformations between stages of active regeneration (anagen), apoptotic involution (catagen), and relative proliferative quiescence (telogen). Due to the low proliferation rate and small size of the HF during telogen, this stage was conventionally thought of as a stage of dormancy. However, multiple lines of newly emerging evidence show that HFs during telogen are anything but dormant. Here, we emphasize that telogen is a highly energy‐efficient default state of the mammalian coat, whose function centres around maintenance of the hair fibre and prompt responses to its loss. While actively retaining hair fibres with minimal energy expenditure, telogen HFs can launch a new regeneration cycle in response to a variety of stimuli originating in their autonomous micro‐environment (including its stem cell niche) as well as in their external tissue macro‐environment. Regenerative responses of telogen HFs change as a function of time and can be divided into two sub‐stages: early ‘refractory’ and late ‘competent’ telogen. These changing activities are reflected in hundreds of dynamically regulated genes in telogen skin, possibly aimed at establishing a fast response‐signalling environment to trauma and other disturbances of skin homeostasis. Furthermore, telogen is an interpreter of circadian output in the timing of anagen initiation and the key stage during which the subsequent organ regeneration (anagen) is actively prepared by suppressing molecular brakes on hair growth while activating pro‐regenerative signals. Thus, telogen may serve as an excellent model system for dissecting signalling and cellular interactions that precede the active ‘regenerative mode’ of tissue remodeling. This revised understanding of telogen biology also points to intriguing new therapeutic avenues in the management of common human hair growth disorders.     

Investigating the developmental onset of regenerative potential in the annelid Capitella teleta

An animal's ability to regrow lost tissues or structures can vary greatly during its life cycle. The annelid Capitella teleta exhibits posterior, but not anterior, regeneration as juveniles and adults. In contrast, embryos display only limited replacement of specific tissues. To investigate when during development individuals of C. teleta become capable of regeneration, we assessed the extent to which larvae can regenerate. We hypothesized that larvae exhibit intermediate regeneration potential and demonstrate some features of juvenile regeneration, but do not successfully replace all lost structures. Both anterior and posterior regeneration potential of larvae were evaluated following amputation. We used several methods to analyze wound sites: EdU incorporation to assess cell proliferation; in situ hybridization to assess stem cell and differentiation marker expression; immunohistochemistry and phalloidin staining to determine presence of neurites and muscle fibers, respectively; and observation to assess re-epithelialization and determine regrowth of structures. Wound healing occurred within 6 h of amputation for both anterior and posterior amputations. Cell proliferation at both wound sites was observed for up to 7 days following amputation. In addition, the stem cell marker vasa was expressed at anterior and posterior wound sites. However, growth of new tissue was observed only in posterior amputations. Neurites from the ventral nerve cord were also observed at posterior wound sites. De novo ash expression in the ectoderm of anterior wound sites indicated neuronal cell specification, although the absence of elav expression indicated an inability to progress to neuronal differentiation. In rare instances, cilia and eyes re-formed. Both amputations induced expanded expression of the myogenesis gene MyoD in preexisting tissues. Our results indicate that amputated larvae complete early, but not late, stages of regeneration, which indicates a gradual acquisition of regenerative ability in C. teleta. Furthermore, amputated larvae can metamorphose into burrowing juveniles, including those missing brain and anterior sensory structures. To our knowledge, this is the first study to assess regenerative potential of annelid larvae.