Mouse Sertoli cells isolation by lineage tracing and sorting

Sertoli cells play a key role in spermatogenesis by supporting the germ cells throughout differentiation. The isolation of Sertoli cells is essential to study their functions. However, the close contact of Sertoli cells with other testicular cell types and the high proliferation of contaminating cells are obstacles to obtain pure primary cultures. Current rodent Sertoli cell isolation protocols result in enriched, rather than pure Sertoli cells. Therefore, novel approaches are necessary to improve the purity of Sertoli cell primary cultures. The goal of this study is to obtain pure mouse Sertoli cells using lineage tracing and fluorescence‐activated cell sorting (FACS). We bred the Amh‐Cre mouse line with tdTomato line to generate mice constitutively expressing red fluorescence specifically in Sertoli cells. Primary cultures of Sertoli cells isolated from prepubertal mice showed that 79% of cells expressed tdTomato, as evaluated by fluorescence microscopy and flow cytometry; however, nearly all adherent cells were positive for vimentin. Most of the tomato‐negative cells expressed α‐smooth muscle actin (α‐SMA), a peritubular myoid cell marker, but double‐negative populations were also present. These findings suggest that vimentin lacks Sertoli cell‐specificity and that α‐SMA is not adequate to identify all of the contaminating cells. Upon FACS sorting; however, virtually 100% of the cells were tdTomato positive, expressed vimentin, but not α‐SMA. Prepubertal mice yielded a higher number of Sertoli cells compared to adults, but both could be adequately sorted. In conclusion, our study shows that lineage tracing and sorting is an efficient strategy for acquiring pure populations of murine Sertoli cells.     

The germ cell lineage identified by vas-mRNA during the embryogenesis in goldfish

vas RNA has been identified in germ-line cells and its precursors in zebrafish, with the result that the germ-line lineage can be traced throughout embryogenesis. In the present study, we described vas localization and the migration of vas-positive cells in goldfish, using whole mount in situ hybridization. The signals of vas mRNA localization appeared at the marginal part of the first to third cleavage planes. The eight signals were detected during the period from the 8- cells to the 512-cell stage. At the late-blastula stage, additional numbers of vas-positive cells were observed, suggesting the proliferation of these cells. At the segmentation period, vas-positive cells showed a long extended distribution along the embryonic axis, but did not form any clusters. vas-positive cells were occasionally distributed at the head region, especially around the future otic vesicle. These signals were inherited to the primordial germ cells, suggesting that vas-positive cells were primordial germ cells (PGCs) in goldfish.     

Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing

Hematopoietic cells have been reported to convert into a number of non-hematopoietic cells types after transplantation/injury. Here, we have used a lineage tracing approach to determine whether hematopoietic plasticity is relevant for the normal development of hepatocytes and endothelial cells, both of which develop in close association with blood cells. Two mouse models were analyzed: vav ancestry mice, in which essentially all hematopoietic cells, including stem cells, irreversibly express yellow fluorescent protein (YFP); and lysozyme ancestry mice, in which all macrophages, as well as a small subset of all other non-myeloid hematopoietic cells, are labeled. Both lines were found to contain YFP+ hepatocytes at similar frequencies, indicating that macrophage to hepatocyte contributions occur in unperturbed mice. However, the YFP+ hepatocytes never formed clusters larger than three cells, suggesting a postnatal origin. In addition, the frequency of these cells was very low (approximately 1 in 75,000) and only increased two- to threefold after acute liver injury. Analysis of the two mouse models revealed no evidence for a hematopoietic origin of endothelial cells, showing that definitive HSCs do not function as hemangioblasts during normal development. Using endothelial cells and hepatocytes as paradigms, our study indicates that hematopoietic cells are tightly restricted in their differentiation potential during mouse embryo development and that hematopoietic plasticity plays at best a minor role in adult organ maintenance and regeneration.     

Colon stem cell and crypt dynamics exposed by cell lineage reconstruction

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems.     

Developmental links between the fetal and adult zones of the adrenal cortex revealed by lineage tracing

The nuclear receptor Ad4BP/SF-1 is essential for development of the adrenal cortex and the gonads, which derive from a common adrenogonadal primordium. The adrenal cortex subsequently forms morphologically distinct compartments: the inner (fetal) and outer (definitive or adult) zones. Despite considerable effort, the mechanisms that mediate the differential development of the adrenal and gonadal primordia and the fetal and adult adrenal cortices remain incompletely understood. We previously identified a fetal adrenal-specific enhancer (FAdE) in the Ad4BP/SF-1 locus that directs transgene expression to the fetal adrenal cortex and demonstrated that this enhancer is autoregulated by Ad4BP/SF-1. We now combine the FAdE with the Cre/loxP system to trace cell lineages in which the FAdE was active at some stage in development. These lineage-tracing studies establish definitively that the adult cortex derives from precursor cells in the fetal cortex in which the FAdE was activated before the organization into two distinct zones. The potential of these fetal adrenocortical cells to enter the pathway that eventuates in cells of the adult cortex disappeared by embryonic day 14.5. Thus, these studies demonstrate a direct link between the fetal and adult cortices involving a transition that must occur before a specific stage of development.     

Theory of cell lineage graphs and their application to the intestinal epithelium

Elucidation of the molecular basis of cell lineage details is a major activity of both developmental biologists and those studying renewing systems in the adult. Given this priority I was surprised to find how little theory has been developed for the main object of this work, the lineage diagram. Even simple questions like—how many different diagrams are plausible?—do not appear to have been addressed and so I decided it would be timely to try to do so here. The results are applied to the intestinal epithelium as an example with special emphasis on the interpretation of recent mutation-based lineages studies.     

Amplified inverted duplications within and adjacent to heterologous selectable DNA.

Plasmids containing a dihydrofolate reductase (DHFR) expression unit were transfected into DHFR-deficient Chinese hamster ovary (CHO) cells. Methotrexate exposure was used to select cells with amplified DHFR sequences. Three cell lines were isolated containing amplified copies of transfected DNA that had integrated into the Chinese hamster genome. Plasmid DNA was found to co-amplify with flanking hamster sequences that were repetitive (2 cell lines) and unique (1 cell line). Fragments comprising the junctions of amplified plasmid and CHO DNA were found to exist as inverted duplications in all three cell lines. These observations provide evidence that inverted duplication occurred prior to DNA amplification, thus underscoring the importance of inverted duplication in the DNA amplification process.     

Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.     

Microenvironmental Heterogeneity and Space Utilization by Desert Vines within their Host Trees

The three-dimensional biomass distribution and the microenvironmentsexperienced by several desert vine species growing within thecanopy of host trees were studied at the Centro Ecológicode Sonora in México. The light environment within thecrown of the host tree Cercidium microphyllum showed a horizontaland vertical gradient from the base of the trunk to the edgesof the canopy. Within this gradient total daily photosyntheticallyactive radiation (PAR) varied from 47.8 mol m^-2outside the crownto 4.6 mol m^-2at the centre of the crown and close to the ground.Maximum air temperature was 3 °C lower beneath the crownthan outside. Within the canopy, most vines experienced lessthan 50% of the daily available PAR outside the canopy. Formost of the day, leaves of vines received 15% or less of themaximum available PAR. Our study shows that vines do not growtowards full sunlight but rather they exploit different habitatpossibilities within their host tree crown. Leaves along thestems of vines experienced a wide range of light environments,showing coefficients of variation (CV) in total daily PAR from36.4 to 94.6%. Daily courses of PAR also showed that leaveswithin the canopy experienced short-term temporal variationin the light environment. Differences in CV of daily PAR valuesand preferences in heterogeneous light microenvironments amongspecies suggested that different vine species might be spatiallyseparated in the canopy. We suggest that in desert habitats,conditions within the crown of host trees result in an importantmicrohabitat that vines can exploit, allowing them to avoidthe high light, temperature and water deficits found in thesurrounding environment. Copyright 1999 Annals of Botany Company Sonoran Desert, vines, host trees, canopy light environment.     

A newly identified locus controls complete resistance to Microcyclus ulei in the Fx2784 rubber clone

Using cultivars which are genetically resistant to South American leaf blight (SALB) caused by the fungus Microcyclus ulei is the only way to plant rubber trees in disease-affected areas. Numerous field observations led to the hypothesis that the resistance of the cultivar Fx2784 to SALB is likely to be monogenic. In this study, we investigated this hypothesis by examining the distribution of the trait in a cross between the resistant cultivar and a susceptible one. The individuals resulting from this cross were planted in field trials in French Guiana and Brazil. The resistance of all the trees was assessed by field observations. Bulk segregant analysis (BSA) using microsatellite markers was performed in French Guiana to determine which markers were genetically linked to resistance, and the results were validated by field observations in Brazil. In both locations, a 1:1 segregation of the resistance trait was observed, thus reinforcing the monogenic hypothesis. BSA showed tight linkage between resistance and the microsatellite markers located in linkage group 2 in the Hevea genome and enabled to pinpoint the resistance locus. The location was confirmed by observations on the trees planted in Brazil. This result should facilitate the use of Fx2784 resistance in future breeding programs for SALB resistance. This is the third major locus conferring resistance to SALB identified in rubber tree (Hevea spp.). These three loci are genetically independent, a favorable situation for genetic improvement of SALB resistance.     

PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration

Background

Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.

Methodology

PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.

Conclusions/Significance

PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior.     

Faecal mutagens and intestinal tumours in the dimethylhydrazine-injected rat

The relationship between the development of colonic tumours and the presence of faecal mutagens has been investigated in the dimethylhydrazine (DMH) injected rat model. Subcutaneous DMH 20 mg/kg for 6 weeks in 10 rats produced colonic tumours in 8 animals and duodenal tumours in 2. Faeces were collected 6, 10, 14, 20 and 25 weeks after commencing DMH injections and were assayed using the Ames test system with both aqueous and ether extracts. All faeces were initially negative for mutagens, but all became positive at some stage following DMH injections, There was no consistent pattern of mutagen excretion or of the strain of Salmonella typhimurium detecting a positive extract. Early positive extracts tended to be with aqueous extracts, whereas later extracts (20 and 25 weeks) were predominantly with ether extracts. DMH produces a metabolic change in the rat resulting in the production of faecal mutagens. This change precedes the appearance of colonic neoplasms and persists after the withdrawal of DMH injections.     

New pockets in dengue virus 2 surface identified by molecular dynamics simulation

One of the factors limiting the search of new compounds based on the structure of target proteins involved in diseases is the limited amount of target structural information. Great advances in the search for lead compounds could be achieved to find new cavities in protein structures that are generated using well established computational chemistry tools. In the case of dengue, the discovery of pockets in the crystallographic structure of the E protein has contributed to the search for lead compounds aimed at interfering in conformational transitions involved in the pH-dependent fusion process. This is a complex mechanism triggered by the acid pH of the endosomes that leads to the initial changes in the E protein assembly at the virus surface. In the present work, an arrangement of three ectodomain portions of the E protein present on the surface of the mature dengue virus was studied through long all-atom molecular dynamics simulations with explicit solvent. In order to identify new pockets and to evaluate the influence of the acid pH on these pockets, the physiological neutral pH conditions and the acid pH of the endosomes that trigger the fusion process were modeled. Several pockets presenting pH-dependent characteristics were found in the contact regions between the chains. Pockets at the protein-protein interfaces induced by a monomer in another monomer were also found. Some of the pockets are good candidates for the design of lead compounds that could interfere in the rearrangements in E proteins along the fusion process contributing to the development of specific inhibitors of the dengue disease.     

Tightly congruent bursts of lineage and phenotypic diversification identified in a continental ant radiation

Adaptive diversification is thought to be shaped by ecological opportunity. A prediction of this ecological process of diversification is that it should result in congruent bursts of lineage and phenotypic diversification, but few studies have found this expected association. Here, we study the relationship between rates of lineage diversification and body size evolution in the turtle ants, a diverse Neotropical clade. Using a near complete, time‐calibrated phylogeny we investigated lineage diversification dynamics and body size disparity through model fitting analyses and estimation of per‐lineage rates of cladogenesis and phenotypic evolution. We identify an exceptionally high degree of congruence between the high rates of lineage and body size diversification in a young clade undergoing renewed diversification in the ecologically distinct Chacoan biogeographical region of South America. It is likely that the region presented turtle ants with novel ecological opportunity, which facilitated a nested burst of diversification and phenotypic evolution within the group. Our results provide a compelling quantitative example of tight congruence between rates of lineage and phenotypic diversification, meeting the key predicted pattern of adaptive diversification shaped by ecological opportunity.