The "third eye"-- a new concept of trans-differentiation of pineal gland into median eye in amphibian tadpoles of Bufo melanostictus

Median third eye was found to develop from transplanted pineal gland of external gill stage tadpoles in the recipient 5 toe stage tadpoles of Bufo melanostictus. Pineal gland along with a bit part of brain tissue of the donor external gill stage tadpole was cut out and transplanted into a pit made between two lateral eyes of 5 toe stage recipient tadpoles. Half of the operated tadpoles were treated with vitamin A (15 IU/ml.) for 15 days. Median "third eye" was found to develop in the both untreated and vitamin A treated tadpoles. However, vitamin A increased the percentage of the development of median eyes. Morphological and histological study revealed that newly transformed median eyes were similar to that of normal functional eyes. A stalk like structure developed which connects the median eye to the brain. The median third eye could not develop when pineal gland of 5 toe stage mature tadpole was transplanted into the tadpole of the same age.     

Fate and protective effect of marrow stromal cells after subretinal transplantation

Engraftment of marrow stromal cells (MSCs) has been proposed as a therapeutic approach for degenerative diseases. In this study we investigated the fate and dynamic progress of grafted MSCs in living retina with the aim of evaluating the use of transplanted MSCs to treat retinal degeneration. Approximately 1×10⁵ gfp -MSCs in 2 μl phosphate-buffered saline were injected into the subretinal space of adult Sprague-Dawley rats. Two weeks later, approximately 0.174%±0.082% of the transplanted cells had survived and diffused into the subretinal space. Nine weeks after transplantation the surviving gfp -MSCs accounted for 0.049%±0.023% of the number of cells injected and were mainly located at the injection site. The same number of MSCs were transplanted into the left eye subretinal space of 3-week-old hereditary retinal degenerative Royal College of Surgeons rats, and phosphate-buffered saline was injected into their right eyes as a control. Five weeks after transplantation, the amount of rudimentary photo-receptors was more significantly increased in grafted eyes than in control eyes. The results indicated that grafted MSCs could survive and rescue retinal degeneration.     

Target-specific innervation by autonomic and sensory nerve fibers in hairy fetal skin transplanted into the anterior eye chamber of adult rat

Summary Pieces of hairy skin tissue of fetal rat were transplanted into the anterior eye chamber of adult rats. The ability of autonomic and sensory nerve fibers from the host iris to innervate the grafted skin tissue was immunohistochemically and enzyme-histochemically examined using antisera against tyrosine hydroxylase (TH), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), and a reaction medium for acetylcholinesterase (AchE). The grafted tissue was successfully implanted and connected with the host iris. Epidermis, dermis, subcutaneous tissue, hairs, hair follicles, sebaceous glands, and piloerector muscles developed in the graft. Two weeks after transplantation, TH-, SP-, and CGRP-immunoreactive fibers were observed in association with the blood vessels in the graft. Four weeks after transplantation, TH-immunoreactive fibers were distributed in the piloerector muscles, whereas SP-and CGRP-immunoreactive fibers were present around the hair follicles. VIP-immunoreactive and AchE-positive fibers were restricted to the host iris at all survival times. These results suggest that the outgrowth of autonomic and sensory nerve fibers from the host iris show target specificity for the grafted skin tissue.     

Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes

In order to determine whether differentiated somatic cells maintain genetic totipotency, nuclear transplantations from several differentiated somatic cell types into eggs and oocytes were performed previously in Rana pipiens and Xenopus laevis. The formation of postneurula embryos and tadpoles under the direction of the test nuclei demonstrated their genetic multipotency. In addition, Rana erythrocyte nuclei transplanted to oocytes directed more extensive tadpole development than those injected into eggs. We have extended our studies of the genomic potential of differentiated somatic nuclei from the peripheral blood of Rana pipiens. First, we show that the developmental potential of erythrocyte nuclei injected into oocytes at first meiotic metaphase was greater than those injected into diplotene oocytes. Second, we demonstrate that erythroblast and leukocyte nuclei transplanted to oocytes at first meiotic metaphase promoted more advanced tadpole development than those previously injected into Xenopus eggs. Third, erythrocyte nuclei were more successful in promoting advanced tadpole development compared with erythroblast and leukocyte nuclei. The results show that differentiated somatic nuclei transferred to the cytoplasm of oocytes at first meiotic metaphase display enhanced genomic and developmental potential over those transplanted to diplotene oocytes and eggs, at least for the three nuclear cell types tested from the peripheral blood.     

Neoblast-enriched fraction rescues eye formation in eye-defective planarian 'menashi' Dugesia ryukyuensis

Planarians are well known for their remarkable regenerative capacity. This capacity to regenerate is thought to be due to the presence of totipotent somatic stem cells known as ‘neoblasts’, which have particular morphological characteristics. The totipotency of neoblasts was supported by Baguñà's experiment, which involved the introduction of donor cells into irradiated hosts. However, since Baguñà's experiment did not include the use of a phenotypic marker, the donor cells could not be traced. In the current study, a genetic mutant planarian, menashi, an eye‐defective mutant that lacks the pigmented area in the eyes, was established. This planarian is excellent for tracing the fate of cells after their introduction into irradiated hosts. To investigate the differentiation potency more directly, a neoblast‐rich fraction obtained from normal worms was transplanted into an X‐ray‐irradiated menashi strain. Planarians that survive X‐ray irradiation were developed, and we observed the pigment of the area in the eyes of the regenerating planarians. This result suggests that the neoblast‐rich fraction contains cells that can proliferate and differentiate. These cells can replace the cells and structures lost by X‐ray irradiation and ablation, and they can also differentiate into eye pigment cells.     

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

胚胎干细胞衍生的视网膜色素上皮细胞移植治疗眼病

Schwartz等报告的用从人胚胎干细胞分化成的视网膜色素上皮细胞（RPE）移植治疗视网膜病,已观察4月,尚属成功。这是首次用从人胚胎干细胞（hESC）定向分化而成的细胞移植至患者取得成功。本文复习RPE移植的历史与现况;hESC分化而成的RPE（hESC-RPE）的实验研究以及临床移植的意义、方法、效果及存在问题,并展望了应用干细胞分化的RPE移植的前景。     

An aberrant retinal pathway and visual centers in Xenopus tadpoles share a common cell surface molecule, A5 antigen

A monoclonal antibody A5 (MAb-A5), which was raised against Xenopus tadpole tectal cells, recognizes a cell surface-related protein molecule (A5 antigen) expressed on the visual centers of Xenopus tadpoles (S. Takagi, T. Tsuji, T. Amagai, T. Takamatsu, and H. Fujisawa, 1987, Dev. Biol. 122, 90-100). The present immunohistochemistry using MAb-A5 indicated that, in addition to the visual centers, A5 antigen was expressed on the general somatic sensory tract in the medulla and spinal cord of Xenopus tadpoles. As the general somatic sensory tract has been shown to be a pathway for ectopically transplanted retinal axons (M. Constantine-Paton and R. R. Capranica, 1976, J. Comp. Neurol. 170, 17-32; M. J. Katz and R. J. Lasek, 1979, J. Comp. Neurol. 183, 817-832), we examined whether retinal axons transplanted close to the spinal cord or medulla preferentially grow into the A5 antigen-positive general somatic sensory tract. We performed eye transplantation at embryonic stages and detected precise locations and trajectories of transplanted retinal axons within the medulla and spinal cord in tadpoles after filling retinal axons with horseradish peroxidase (HRP). HRP histochemistry in combination with MAb-A5 immunohistochemistry indicated that almost all HRP-filled transplanted retinal axons joined the A5 antigen-positive general somatic sensory tract. These findings suggest the involvement of A5 antigen in specific cell-cell recognition between retinal axons and their targets.     

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.     

The structure of oestrogen sensitized rat vagina after exposure to steroids as studied in transplants

Summary Grafts from the vagina and the uterus were transplanted to the anterior chamber of the eye together with crystals of different steroids to investigate whether either the grafted vagina or uterus could be used as indicator of locally produced gestagens.Autologous vaginal and uterine tissues were transplanted into the anterior chamber of the eyes of 199 adult spayed female rats. Crystals of various steroids were placed near to the transplant of the right eye to investigate the local reaction. The steroids used were oestradiol-17, androst-4-ene-317 dione, testosterone, testosterone acetate, progesterone and 3-hydroxypregn-5-en-20-one. The transplant in the left eye served as a control for general reactions. Most of the animals were injected with oestradiol benzoate in order to sensitize the vagina and uterus.The daily injections with 0.1 g oestradiol benzoate produced a typical oestrous picture with a cornified epithelium both in the vagina in situ and in the grafts. The oestrous reaction increased when oestradiol crystals were implanted or when higher doses of oestradiol benzoate were injected.When testosterone or testosterone acetate was implanted in the eye the epithelium of the vaginal transplant was thinner than when only oestrogen had been given.When androsteredione crystals were used, the epithelium of the transplanted vagina was either a rather thick, non-cornified epithelium or a stratified squamous epithelium. In both epithelia mucus could be found in the cells in the upper-most layers.Implantation of progesterone changed the oestrous picture. The epithelium consisted of only 2–3 layers of cells. The upper-most layer or layers presented small to large swollen columnar mucin cells.When pregnenolone crystals were used, the grafted vagina reacted as in the progesterone series though more weakly.The uterus was not suitable for transplantation as it mostly changed into a cystic undifferentiated structure.The vagina in situ and the vaginal graft in the left eye which was not supplied with a steroid showed no characteristics that were marked and constant for any of the steroids except for the oestrogen.In the uterus in situ the gestagens, especially progesterone, caused a stimulation of the stromal cells. Their nuclei became vesicular and showed fine chromatin particles.The investigation was supported by grants from the Medical Faculty of Lund and The Royal Physiographic Society of Lund.     

Transplanted neurons form both normal and ectopic projections in the adult brain

Transplantation of embryonic or stem cell derived neurons has been proposed as a potential therapy for several neurological diseases. Previous studies reported that transplanted embryonic neurons extended long-distance projections through the adult brain exclusively to appropriate targets. We transplanted E14 lateral ganglionic eminence (LGE) and E15 cortical precursors from embryonic mice into the intact adult brain and analyzed the projections formed by transplanted neurons. In contrast to previous studies, we found that transplanted embryonic neurons formed distinct long-distance projections to both appropriate and ectopic targets. LGE neurons transplanted into the adult striatum formed projections not only to the substantia nigra, a normal target, but also to the claustrum and through all layers of fronto-orbital cortex, regions that do not normally receive striatal input. In some cases, inappropriate projections outnumbered appropriate projections. To examine the relationship between the donor cells and host brain in establishing the pattern of projections, we transplanted cortical precursors into the adult striatum. Despite their heterotopic location, cortical precursors not only predominantly formed projections appropriate for cortical neurons, but they also formed projections to inappropriate targets. Transplantation of GFP-expressing cells into beta-galactosidase-expressing mice confirmed that the axonal projections were not created by the fusion of donor and host cells. These results suggest that repairing the brain using transplantation may be more complicated than previously expected, because exuberant ectopic projections could result in brain dysfunction. Understanding the signals regulating axonal extension in the adult brain will be necessary to harness stem cells or embryonic neurons for effective neuronal-replacement therapies.     

Cell Migration and Induction in the Development of the Surface Ectodermal Pattern of the Xenopus laevis Tadpole

The surface of the Xenopus tadpole contains three specialized, transient cell types; the ciliated, hatching gland, and cement gland cells. To distinguish whether the appearance of these cell types on the surface is due to induction of surface cells or due to migration of deep ectodermal cells into the surface, we transplanted labelled surface or deep cells to unlabelled hosts at early to mid-gastrulae. After raising the host to a tadpole (Stage 28), we examined the embryo's surface for ciliated, hatching gland, and cement gland cells, and observed which cells were labelled. We find that all ciliated cells move into the surface from the deep ectodermal layer along with other cells of unknown function. Hatching gland cells arise by induction of surface cells as do the majority of cement gland cells. A few deep cells give rise to cement gland cells. Therefore, migration of deep cells to the surface and localized induction of surface cells contribute to the final surface patterning of the Xenopus tadpole.     

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.     

绿色荧光蛋白基因修饰的人视网膜色素上皮细胞异种移植的长期观察

探讨利用视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞移植介导目的基因转移至视网膜的可行性。人的RPE细胞在体外经携带绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因的逆转录病毒感染及G4 1 8筛选后 ,手术显微镜直视下经睫状体平坦部注射到兔眼视网膜下间隙。术后通过活体荧光眼底照相 ,荧光显微镜、共聚焦显微镜及透射电镜等观察眼球铺片及切片 ,发现经 gfp基因修饰的人RPE细胞在兔眼视网膜下间隙可存活一年以上。移植的RPE gfp细胞不仅仅局限于移植部位 ,大多扩散到超过 2～ 3个象限的眼底 ,镶嵌于宿主RPE细胞之间或呈单层排列在宿主色素上皮与神经视网膜之间 ,并持续高水平表达GFP。移植后早期玻璃体内每周注射免疫抑制剂普乐可复 (FK5 0 6 )可明显改善移植细胞的存活状态     

Region-specific generation of functional neurons from naive embryonic stem cells in adult brain

Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into functional neurons when transplanted in the central nervous system. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipient tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders.     

Neural stem cells transplanted into intact brains as neurospheres form solid grafts composed of neurons, astrocytes and oligodendrocyte precursors

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.     

Modification of the proliferative capacity of transplanted bone marrow colony forming units by changes in the host environment

Regulation of the proliferation of transplanted colony forming units (CFUs) was investigated in lethally irradiated mice, pretreated by methods known to accelerate hemopoietic recovery after sublethal irradiation. Prospective recipients were exposed to either hypoxia, vinblastine or priming irradiation and at different intervals thereafter lethally irradiated and transplanted with bone marrow. Repopulation of CFUs was determined by counting the number of splenic colonies in primary recipients or by retransplantation. Regeneration of grafted CFUs was greatly accelerated and their self-renewal capacity increased in mice grafted within two days after hypoxia. Also the number of splenic colonies formed by grafted syngeneic CFUs as well as by C57BL parent CFUs growing in BC3F1 hosts was significantly increased. The effect was not dependent on the seeding efficiency of CFUs and apparently resulted from hypoxia induced changes in the hosts physiological environment. Proliferative capacity of grafted CFUs increased remarkably in hosts receiving vinblastine two or four days prior to irradiation. Priming irradiation given six days before main irradiation accelerated, given two days before impaired regeneration of CFUs. The increased rate of regeneration was not related to the cellularity of hemopoietic organs at the time of transplantation. The growth of CFUs in diffusion chambers implanted into posthypoxic mice was only slightly improved which does indicate that the accelerated regeneration of CFUs in posthypoxic mice is mainly due to the changes in the hemopoietic microenvironment. A short conditioning of transplanted CFUs by host factor(s) was sufficient to improve regeneration. The results might suggest that the speed of hemopoietic regeneration depends on the number of CFUs being induced to proliferate shordy after irradiation, rather than on the absolute numbers of CFUs available to the organism.     

The DNase domain-containing protein TATDN1 plays an important role in chromosomal segregation and cell cycle progression during zebrafish eye development

The DNase domain-containing protein TATDN1 is a conserved nuclease in both prokaryotes and eukaryotes. It was previously implicated to play a role in apoptotic DNA fragmentation in yeast and C. elegans. However, its biological function in higher organisms, such as vertebrates, is unknown. Here, we report that zebrafish TATDN1 (zTATDN1) possesses a novel endonuclease activity, which first makes a nick at the DNA duplex and subsequently converts the nick into a DNA double-strand break in vitro. This biochemical property allows zTATDN1 to catalyze decatenation of catenated kinetoplast DNA to produce separated linear DNA in vitro. We further determine that zTATDN1 is predominantly expressed in eye cells during embryonic development. Knockdown of TATDN1 in zebrafish embryos results in an abnormal cell cycle progression, formation of polyploidy and aberrant chromatin structures. Consequently, the TATDN1-deficient morphants have disordered eye cell layers and significantly smaller eyes compared with the WT control. Altogether, our current studies suggest that zTATDN1 plays an important role in chromosome segregation and eye development in zebrafish.     

The DNase domain-containing protein TATDN1 plays an important role in chromosomal segregation and cell cycle progression during zebrafish eye development

The DNase domain-containing protein TATDN1 is a conserved nuclease in both prokaryotes and eukaryotes. It was previously implicated to play a role in apoptotic DNA fragmentation in yeast and C. elegans. However, its biological function in higher organisms, such as vertebrates, is unknown. Here, we report that zebrafish TATDN1 (zTATDN1) possesses a novel endonuclease activity, which first makes a nick at the DNA duplex and subsequently converts the nick into a DNA double-strand break in vitro. This biochemical property allows zTATDN1 to catalyze decatenation of catenated kinetoplast DNA to produce separated linear DNA in vitro. We further determine that zTATDN1 is predominantly expressed in eye cells during embryonic development. Knockdown of TATDN1 in zebrafish embryos results in an abnormal cell cycle progression, formation of polyploidy and aberrant chromatin structures. Consequently, the TATDN1-deficient morphants have disordered eye cell layers and significantly smaller eyes compared with the WT control. Altogether, our current studies suggest that zTATDN1 plays an important role in chromosome segregation and eye development in zebrafish.