Generation of mouse-zebrafish hematopoietic tissue chimeric embryos for hematopoiesis and host-pathogen interaction studies.

Xenografts of the hematopoietic system are extremely useful as disease models and for translational research. Zebrafish xenografts have been widely used to monitor blood cancer cell dissemination and homing due to the optical clarity of embryos and larvae, which allow unrestricted in vivo visualization of migratory events. Here, we have developed a xenotransplantation technique that transiently generates hundreds of hematopoietic tissue chimeric embryos by transplanting murine bone marrow cells into zebrafish blastulae. In contrast to previous methods, this procedure allows mammalian cell integration into the fish developmental hematopoietic program, which results in chimeric animals containing distinct phenotypes of murine blood cells in both circulation and the hematopoietic niche. Murine cells in chimeric animals express antigens related to (i) hematopoietic stem and progenitor cells, (ii) active cell proliferation and (iii) myeloid cell lineages. We verified the utility of this method by monitoring zebrafish chimeras during development using in vivo non-invasive imaging to show novel murine cell behaviors, such as homing to primitive and definitive hematopoietic tissues, dynamic hematopoietic cell and hematopoietic niche interactions, and response to bacterial infection. Overall, transplantation into the zebrafish blastula provides a useful method that simplifies the generation of numerous chimeric animals and expands the range of murine cell behaviors that can be studied in zebrafish chimeras. In addition, integration of murine cells into the host hematopoietic system during development suggests highly conserved molecular mechanisms of hematopoiesis between zebrafish and mammals.This article has an associated First Person interview with the first author of the paper.

Generation of Viable Plant-Vertebrate Chimeras.

The extreme dependence on external oxygen supply observed in animals causes major clinical problems and several diseases are related to low oxygen tension in tissues. The vast majority of the animals do not produce oxygen but a few exceptions have shown that photosynthetic capacity is physiologically compatible with animal life. Such symbiotic photosynthetic relationships are restricted to a few aquatic invertebrates. In this work we aimed to explore if we could create a chimerical organism by incorporating photosynthetic eukaryotic cells into a vertebrate animal model. Here, the microalgae Chlamydomonas reinhardtii was injected into zebrafish eggs and the interaction and viability of both organisms were studied. Results show that microalgae were distributed into different tissues, forming a fish-alga chimera organism for a prolonged period of time. In addition, microscopic observation of injected algae, in vivo expression of their mRNA and re-growth of the algae ex vivo suggests that they survived to the developmental process, living for several days after injection. Moreover microalgae did not trigger a significant inflammatory response in the fish. This work provides additional evidence to support the possibility that photosynthetic vertebrates can be engineered.

Production of chimeric embryos by aggregation of bovine egfp eight-cell stage blastomeres with two-cell fused and asynchronic embryos.

Embryo disaggregation allows the production of two to four identical offspring from a single cow embryo. In addition, embryo complementation has become the technique of choice to demonstrate the totipotency of embryonic stem cells and induced pluripotent stem cells. Therefore, the aim of this study was to generate a new and simple method by aggregation in the well-of-the-well system to direct each single enhanced green fluorescent protein (egfp) eight-cell blastomere derived from bovine in vitro fertilization embryos to the inner cell mass (ICM) of chimeras produced with fused and asynchronic embryos. To this end, the best conditions to generate in vitro fertilization-fused embryos were determined. Then, the fused (F) and nonfused (NF) embryos were aggregated in two distinct conditions: synchronically (S), with both transgenic and F embryos produced on the same day, and asynchronically (AS), with transgenic embryos produced one day before F embryos. The highest fusion and blastocysts rates were obtained with two pulses of 40 V. The 2ASF and 2ASNF groups showed the best number of blastocysts expressing the EGFP protein (48% and 41%, respectively). Furthermore, the 2ASF group induced the highest localization rates of the egfp-expressing blastomere in the ICM (6/13, 46% of ICM transgene-expressing blastocysts). This technique will have great application for multiplication of embryos of high genetic value or transgenic embryos and also with the generation of truly bovine embryonic stem cells and induced pluripotent stem cells.

Apomixis in different ploidy levels of cassava.

Two polyploid hybrids between cassava (Manihot esculenta) cultivar 307-2 and its wild relatives M. glaziovii and M. anomala, were studied to examine the relationship between ploidy level and the production of seeds without fertilization. A clearing method was applied to assess ovule sizes as an indication of multiembryony. The diploid cultivar 307-2 had regular 18 bivalents at meiotic metaphase 1 while the polyploid types showed chromosome configurations varying from 3 to 4 quadrivalents and 28 to 30 bivalents. A total of 14% of studied ovules of the polyploid hybrid involving M. glaziovii were multiebryonic, while the percentage of multiembryony was as low as 2% in the polyploid hybrid M. anomala×M. esculenta. Diploid hybrid types did not show any multi embryony. Adventitious embryos were found and documented for the first time in polyploid hybrids M. esculenta×M. glaziovii. The association of multiple embryo formation with ovary size and pollination showed that apomictic embryos form independently from fertilization. Simple iodized carmine stain for measuring pollen viability proved as efficient as the sophisticated Alexander method.

[RNA/DNA ratio as an index of physiological condition of Colossoma macropomum and Piaractus branchypomus (Pisces: Characiformes) during embryonic development]. / Relación ARN/ADN como indice de condición fisiológica del híbrido de la cachama Colossoma macropomum y el morocoto piaractus brachypomus durante el desarrollo embrionario.

We evaluated RNA/DNA ratio as an index of physiological condition during larval development of a hybrid between the fishes Colossoma macropomum (cachama) and Piaractus brachypomus (morocoto). The samples were obtained by induced reproductive technology and the eggs were maintained in acrylic conical incubator with a continuous waterflow. Embryonic development, from egg fertilization to cell division and hatch out, took 12 hours 20 minutes at 29.5 degrees C, dissolved oxygen contents of 6.0 ppm and pH 7.5. Nucleic acids quantification was determined by fluorometry with ethidium bromide and Hoechst 33258 dyes. We observed significant changes of RNA/DNA ratios during all stages of the embryonic larval development. Therefore, RNA/DNA relation is an useful technique to evaluate physiological condition in short period and could be utilized as nutritional condition and/or instantaneous growth for routine check to verify the health status in early life of cultivated species.