Effect of conspecifics on sexual maturation in female European pine voles (Pitymys subterraneus)

Using the number of large ovarian follicles (Type 8) as an indicator of sexual maturation we found that urinary compounds released by adult males accelerated puberty while urine from females suppressed hormonal activity in juvenile female European pine voles. The release of chemosignals that delayed puberty of juvenile females was not influenced by ovarian hormones; urine from ovariectomized females was as effective as urine from unoperated animals.     

Variation in the growth and development of the hind limbs in frogs of the genus Telmatobius (Anura: Telmatobiidae)

There are remarkable interspecific differences in the sizes of the larvae of Andean frogs of the genus Telmatobius. This size variation seems to be associated with the duration of the larval stage and may affect the hind-limb morphology in Telmatobius. Larval, juvenile, and adult Telmatobius rubigo and T. oxycephalus were examined to determine the variation in relative sizes of hind-limb elements, their growth patterns during postmetamorphic life, and skeletal ontogeny. The results showed that the proportionately shorter hind limbs of T. rubigo relative to those of T. oxycephalus are associated with the protracted development and ossification of hind limbs during the prolonged larval life of T. rubigo. Postmetamorphically, the hind limbs grew faster than the body in juveniles of both species in contrast to the relative growth rates of the hind limbs and bodies of the adults. The growth phase of juvenile T. rubigo seems shorter than that of juvenile T. oxycephalus; possibly, this heightens the difference in the relative lengths of hind limbs after metamorphosis. Temperature affects the effects of thyroid hormone on growth and development, and T. rubigo lives at much higher, colder elevations than does T. oxycephalus. It is not clear whether the developmental differences described here are plastic (i.e., environmentally induced) or genetically fixed in each species.     

Identification of a gene involved in the juvenile-to-adult transition (JAT) in cultivated olive trees

The juvenile-to-adult transition is a complex and poorly understood process in plant development required to reach reproductive competence. For woody plants, knowledge of this transition is even scantier and no genes have been definitively identified as involved in this transition. To search for genes involved in the juvenile-to-adult transition in olive, we constructed juvenile and adult subtractive cDNA gene libraries and identified genes that were differentially expressed in the juvenile and adult phases. In the analysis of theses libraries, we found 13 differentially expressed genes. One of these genes designated as juvenile to adult transition (JAT) was of special interest because it was highly expressed at the mRNA level in the early developmental phases but repressed in the adult phase. The analysis of mutant trees altered in the juvenile-to-adult transition, as well as a segregating progeny of 31 trees from a “Picual” x “Jabaluna” cross, support the contention that its activity might be required for a non-delayed transition. The study of an Arabidopsis thaliana JAT mutant strain confirmed this hypothesis as it showed a delayed flowering phenotype. JAT is expressed in different parts of the plant, showing an unexpectedly high level of mRNA in the roots. However, the JAT expression level is not determined by the distance to the roots, but rather depends on the developmental stage of the branch meristems. JAT is a widely represented gene in plants that appears to be involved in the control of the juvenile-to-adult transition in olive.     

Quantifying the frequency of tumor-propagating cells using limiting dilution cell transplantation in syngeneic zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency. Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.     

Fetal intestinal transplants in syngeneic rats: a developmental model of intestinal immunity

We conducted a longitudinal study of the development of lymphoid tissue in fetal small intestine transplanted to a subcutaneous site in adult syngeneic Fischer strain rats. Fetal jejunoileal segments obtained between 18 and 21 days of gestation were transplanted to a dorsal subcutaneous site on syngeneic adult rats. Three weeks later, intestinal segments greater than 2.5 cm in length were found in 70% of recipients. Each week for 6 wk post-transplantation, a full-thickness biopsy was obtained for histologic and immunohistologic examination. At the time of transplantation, fetal rat intestine did not display Peyer's patches, intraepithelial lymphocytes, lymphoid follicles, or IgA-containing plasma cells. These lymphoid structures reached adult levels by 4 wk after transplantation, and the sequence of development of the lymphoid structures in the transplants appeared to match the postnatal development of normal small intestine. After immunizing the in situ intestine or the transplanted fetal intestine with cholera toxin, the number of cells producing specific antibodies to the immunogen increased significantly in intestinal transplants and in situ intestine. In contrast, few if any cells synthesizing antibodies to cholera toxin developed in the transplants after i.p. immunization. This study suggests that fetal intestinal transplants behave as part of the mucosal immune system. This model may provide useful approaches to studying the development of mucosal immunity.     

The role of miR156 in developmental transitions in Nicotiana tabacum

Plants undergo a series of developmental transitions during their life cycle. After seed germination, plants pass through two distinct phases: the vegetative phase in which leaves are produced and the reproductive phase in which flowering occurs. Based on the reproductive competence and morphological changes, the vegetative phase can be further divided into juvenile and adult phases. Here, we demonstrate that the difference between juvenile and adult phase of Nicotiana tabacum is characterized by the changes in leaf size, leaf shape as well as the number of leaf epidermal hairs(trichomes). We further show that miR156, an age-regulated microR NA, regulates juvenile-to-adult phase transition in N. tabacum. Overexpression of miR156 results in delayed juvenile-to-adult transition and flowering. Together, our results support an evolutionarily conserved role of miR156 in plant developmental transitions.     

Central insulin sensitivity in male and female juvenile rats

The incidence of juvenile obesity is increasing at an alarming rate. In adults, central insulin administration decreases hypothalamic orexigenic neuropeptides, food intake and body weight more effectively in males than females. Mechanisms regulating energy balance in juvenile animals are inherently different from those in adults due to differences in growth rates and hormonal milieu. Therefore, we sought to determine if central insulin treatment in juvenile rats (4 wk) would have similar sex-dependent effects on food intake as those reported in adult rats. Twenty-four hour food intake was measured following icv saline or insulin (0.01 or 0.1 U) prior to the onset of dark phase of the light cycle. An additional set of animals was used to assess the effects of central insulin on hypothalamic orexigenic (NPY, AgRP) and anorexigenic (POMC) neuropeptide mRNA expression. In both males and females, insulin reduced meal size initially (first 4 h) and later decreased meal frequency (4-24 h) to reduce cumulative food intake. Consistent with this, central insulin decreased hypothalamic NPY and AgRP and increased POMC mRNA expression. In contrast to adult studies, there were no demonstrated sex differences. These studies indicate that juvenile females and males are equally sensitive to central insulin anorexigenic effects, perhaps due to a lack of circulating gonadal hormones. The anorexigenic responsiveness of both genders suggests a potential pharmacologic approach to childhood obesity.     

施氏鲟仔鱼发育圾异速生长模型

施氏鲟仔鱼的生长发育可分为两个时期:卵黄囊期(或称为自由胚期),即从刚出膜(0日龄,10.17±0.63 mm)到初次开口(9日龄,18.93±0.74 mm);晚期,从开口摄食至器官发育基本完全(38日龄,41.89±5.09 mm).卵黄囊期仔鱼的感觉、摄食、呼吸、游泳等器官快速分化;晚期仔鱼各骨板分化并发育,在形态上逐渐完成向成鱼的转变.对施氏鲟仔鱼异速生长进行的研究表明,仔鱼许多关键器官均存在异速生长现象,如眼径、口宽、尾鳍长、胸鳍长分别在2日龄、8~9日龄、10日龄、11日龄出现生长拐点,拐点之前器官快速生长,拐点之后生长速度减慢甚至近似等速生长.施氏鲟仔鱼各器官呈现出协调和快速发育的特征,随着重要的感觉、摄食、呼吸、游泳等器官的发育和完善,仔鱼快速地具备了躲避敌害和摄食的能力,其生存能力大大提高.     

Hepatic proliferation in Gunn rats transplanted with hepatocytes: effect of retrorsine and tri-iodothyronine

Hepatocyte transplantation would offer an attractive alternative to liver transplantation in the treatment of inborn errors of liver metabolism. However, a major problem in most transplantation studies to date has been the limited growth of transplanted cells in the recipient organ. We performed a strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting liver cells in conjunction with repeated administration of triiodothyronine, an inducer of hepatocyte proliferation in rats. In the present study, foetal and adult syngeneic hepatocyte transplantation into spleen was performed in retrorsine-treated hyperbilirubinemic Gunn rats. In parallel, repeated injections of triiodothyronine were given to recipients. Rats were sacrificed at 1, 7, 30 and 90 days after transplantation and blood and bile samples were taken to assess the functionality of transplanted cells. The proliferative activity of transplanted hepatocytes was evaluated using proliferating cell nuclear antigen labelling index. In summary, both adult and foetal hepatocyte transplantation were effective in correcting a metabolic abnormality in Gunn rats for as long as 3 months. The RS/T3 model, as a measure to increase graft function, could represent an important advance to future clinical application of hepatocyte transplantation.     

The Role of Programmed Cell Death Ligand-1 (PD-L1/CD274) in the Development of Graft versus Host Disease

Programmed cell death ligand-1 (PD-L1/CD274) is an immunomodulatory molecule involved in cancer and complications of bone marrow transplantation, such as graft rejection and graft-versus-host disease. The present study was designed to assess the dynamic expression of this molecule after hematopoietic stem cell transplantation in relation to acute graft-versus-host disease. Female BALB/c mice were conditioned with busulfan and cyclophosphamide and transplanted with either syngeneic or allogeneic (male C57BL/6 mice) bone marrow and splenic cells. The expression of PD-L1 was evaluated at different time points employing qPCR, western blot and immunohistochemistry. Allogeneic- but not syngeneic-transplanted animals exhibited a marked up-regulation of PD-L1 expression in the muscle and kidney, but not the liver, at days 5 and 7 post transplantation. In mice transplanted with allogeneic bone marrow cells, the enhanced expression of PD-L1 was associated with high serum levels of IFNγ and TNFα at corresponding intervals. Our findings demonstrate that PD-L1 is differently induced and expressed after allogeneic transplantation than it is after syngeneic transplantation, and that it is in favor of target rather than non-target organs at the early stages of acute graft-versus-host disease. This is the first study to correlate the dynamics of PD-L1 at the gene-, protein- and activity levels with the early development of acute graft-versus-host disease. Our results suggest that the higher expression of PD-L1 in the muscle and kidney (non-target tissues) plays a protective role in skeletal muscle during acute graft-versus-host disease.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.     

FURTHER STUDY RE-EXAMINING WHETHER LARVAL EPIDERMIS CAN OMIT THE SECRETION OF A PUPAL CUTICLE, USING THE SILKWORM, BOMBYX MORI

When larval tissue is exposed to a hormonal milieu lacking juvenile hormone, adult characters appear directly, omitting the pupal stage, in some insects but not in others, including Bombyx mori. An attempt was made to induce omission of pupal characters in this species by varying the stage of the larval epidermis to be tested. Pieces of larval integument taken from fourth- and fifth-instar larvae of various stages were transplanted to developing adults. Although the number of cuticle layers and the types of cuticle produced differed depending on the age of the donors, none of the pieces omitted secreting the pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle, and that a transition of tissue competence may play an important part in the sequential appearance of larval, pupal, and adult characters.     

Features of Transition from Larva to Juvenile in Fishes with Different Types of Early Ontogeny

The transition from the larva state to the juvenile state (i.e. morphological condition characterised by mainly adult characters) was examined in three marine fish species: herring Clupea pallasi marisalbi, wolffish Anarhichas lupus, and eelpout Zoarces viviparus, based on external morphology and skeletal development. In spite of the different reproductive styles (oviparity, facultative viviparity, and obligate viviparity, respectively) and different types of early ontogeny (indirect, transitory, and direct, respectively), the beginning of the juvenile state occurred at similar total lengths (TL), which were approximated as 35 mm TL in herring and eelpout, and 32 mm TL in wolffish. Features of ontogeny were compared, assuming that the beginning of the juvenile state represented an uniform characteristic of morphological development for these species. It was proposed that the beginning of the larva or juvenile periods (sensu Balon) could not coincide with the beginning of the larva and juvenile states in the ontogeny of some species.     

Histochemical characteristics of soleus muscle in hGH transgenic mice

Histochemical characteristics of soleus muscle were compared in human growth hormone (hGH) transgenic mice vs their nontransgenic littermates. Plasma of transgenic mice contained hGH (7.1 +/- 0.7 and 6.7 +/- 0.4 ng/ml, mean +/- SE, at 5 and 11 months of age, respectively); hGH was not detectable in plasma of nontransgenic littermates. Body and soleus weights were greater (approximately 55 and 25%, respectively) and both type I and type IIA fibers were larger in transgenic animals. Most significantly, fiber type composition of the soleus muscle was different in hGH-transgenic animals, i.e., the percentage of type I fibers was significantly greater than in nontransgenic mice (77.2 +/- 5.1% vs 58.4 +/- 2.5%). It is generally believed that skeletal muscle fiber composition is determined predominantly by neural influences (1, 2). These data suggest hormonal factors, growth hormone, also affect the phenotype of skeletal muscle myosin.     

miRNA control of vegetative phase change in trees

After germination, plants enter juvenile vegetative phase and then transition to an adult vegetative phase before producing reproductive structures. The character and timing of the juvenile-to-adult transition vary widely between species. In annual plants, this transition occurs soon after germination and usually involves relatively minor morphological changes, whereas in trees and other perennial woody plants it occurs after months or years and can involve major changes in shoot architecture. Whether this transition is controlled by the same mechanism in annual and perennial plants is unknown. In the annual forb Arabidopsis thaliana and in maize (Zea mays), vegetative phase change is controlled by the sequential activity of microRNAs miR156 and miR172. miR156 is highly abundant in seedlings and decreases during the juvenile-to-adult transition, while miR172 has an opposite expression pattern. We observed similar changes in the expression of these genes in woody species with highly differentiated, well-characterized juvenile and adult phases (Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima), as well as in the tree Populus x canadensis, where vegetative phase change is marked by relatively minor changes in leaf morphology and internode length. Overexpression of miR156 in transgenic P. x canadensis reduced the expression of miR156-targeted SPL genes and miR172, and it drastically prolonged the juvenile phase. Our results indicate that miR156 is an evolutionarily conserved regulator of vegetative phase change in both annual herbaceous plants and perennial trees.     

Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.     

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.Download video file.^{(90M, mov)}     

Fiber type composition of unoperated rat soleus and extensor digitorum longus muscles after unilateral isotransplantation of a foreign muscle in long-term experiments

We examined the effects of the unilateral heterochronous isotransplantation on the fiber type composition and myosin heavy chain (MyHC) isoform content of unoperated slow soleus and fast extensor digitorum longus muscles of female inbred Lewis strain rats. Comparison was made between "control" unoperated muscles of experimental rats (after intramuscular transplantation surgery) with the corresponding muscles of completely naive (unoperated) rats of three age groups (5-, 8- and 14-month-old). This was done in order to ascertain whether these muscles can be used as reliable controls to the transplanted and host muscles for our ongoing grafting experiments. The fiber type composition was determined by assessing the histochemical reaction for myofibrillar adenosine triphosphatase, the MyHC isoform content was determined immunocytochemically using monoclonal antibodies specific to different MyHC isoforms and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Our experiments show that the heterochronous intramuscular isotransplantation procedure had no significant effect on the fiber type composition and MyHC isoform content of the "control" unoperated muscles of the experimental rats when compared to the corresponding muscles of the naive animals. Furthermore, the duration and type of isotransplantation did not also lead to differences among corresponding "control" muscles of experimental animals. We conclude that the unoperated muscles of the experimental rats can be used as controls in our current transplantation project dealing with long-term grafting experiments.     

Morphological and physiological changes,and the functional analysis of <Emphasis Type="Italic">PdSPL9</Emphasis> in the juvenile-to-adult phase transition of <Emphasis Type="Italic">paeonia delavayi</Emphasis>

Tree peony has a long juvenile stage, which has limited breeding efforts. To date, very little information is available regarding the juvenile stage of tree peony. In the present study, Paeonia delavayi plants of varying ages were used to investigate the juvenile phase, juvenile-to-adult phase transition, and adult phase via morphological, physiological, and molecular genetic analysis. Micro-observation of buds of different ages and flower induction experiments suggested that the juvenile-to-adult phase transition of P. delavayi occurred at around 2-years of age. Plant height, crown width, leaf length, and the soluble sugar and starch content were positively correlated with plant age. The juvenile gene PdSPL9 contained miR156 targeted sites and a typical SBP domain was cloned from P. delavayi. Expression analysis showed that the expression levels of PdSPL9, PdmiR172d, and PdLFY increased with plant age, while the inverse pattern was observed for PdmiR156a. Function of PdSPL9 was further characterized in Arabidopsis plant. According to function characterization of PdSPL9 in Arabidopsis and expression patterns of PdSPL9 in P. delavayi, it was suggested that PdSPL9 plays a role in controlling juvenile-to-adult phase transition by promoting miR172d, and PdLFY expression in P. delavayi plants, while the expression of PdSPL9 is repressed by miR156a.     

Glossy15 Controls the Epidermal Juvenile-to-Adult Phase Transition in Maize

Loss-of-function mutations at the maize Glossy15 (Gl15) locus alter the normal transition from juvenile-to-adult growth by conditioning the abbreviated expression of juvenile epidermal cell traits and the coordinate precocious expression of adult epidermal cell features. These include epicuticular wax composition, cell wall characteristics, and the presence or absence of differentiated epidermal cell types (e.g., epidermal macrohairs and bulliform cells). A transposon-induced mutable allele of Glossy15 (gl15-m1) was isolated and employed in both phenotypic and genetic analyses to characterize the role of Gl15 in the maize juvenile-to-adult phase transition. Comparisons between Gl15-active and Gl15-inactive somatic sectors in the leaves of variegated plants demonstrated that the Gl15 gene product acts in a cell-autonomous manner to direct juvenile epidermal differentiation but does not affect factors that regulate the overall process of phase change. Examination of the gl15-m1 phenotype in the Corngrass1, Teopod1, and Teopod2 mutant backgrounds showed that the prolonged expression of juvenile epidermal traits associated with these mutations also required Gl15 activity. These results support a model whereby the cell-autonomous Gl15 gene product responds to a juvenility program that operates throughout the vegetative shoot to condition the juvenile differentiation of maize leaf epidermal cells.