And Immune Systems of the Avian and Mammalian Embryo

Fran oise Dieterlen-Li vre, Sophie Creuzet, and Josselyne Salaun In vivo experimental approaches that have been designed to study the ontogeny of the hematopoietic system in higher vertebrates are described in the present chapter. The avian embryo is directly available to manipulations in ovo during gastrulation and organogenesis. This permissiveness has led to the design of various approaches that provided crucial insights into the ontogeny of the hematopoietic system, particularly regarding...

Injection

Busulfan Administration to Pregnant Dams see Notes 2-4 1. Timed pregnant C57Bl6 J CD45.2 expressing mice are bred to give birth at approx midnight before the day of anticipated transplantation studies. We find that our colony of C57BL6 J mice give birth 19.5 d after coitus and thus plan on performing the transplant on d 20 after coitus. 2. On the morning of d E17 and E18, the pregnant dam is given an intraperitoneal injection of 15.5 mg kg of busulfan see Note 3 . Each dose is delivered...

Expansion and Differentiation of Immature Mouse and Human Hematopoietic Progenitors

Helmut Dolznig, Andrea Kolbus, Cornelia Leberbauer, Uwe Schmidt, Eva-Maria Deiner, Ernst W. M llner, and Hartmut Beug A prerequisite for proper investigation of self-renewal and differentiation of hematopoietic cells is the possibility to obtain large quantities of homogenous primary progenitors under defined conditions, allowing meaningful biochemical and molecular analyses. These cells should show renewal and differentiation characteristics similar to the in vivo situation. The serum-free...

In Situ Hybridization With Cryostat Sections and Whole Mouse Embryos

Riboprobe Synthesis and Purification 1. Mix linearized template deoxyribonucleic acid DNA equivalent to 1 g with probe in labeling solution. 2. Incubate mixture at 37 C for 2-3 h for in vitro transcription. 3. Add 2 L of DNAse I and 1 L of RNAsin to the reaction tube, place on ice, and then spin down, to terminate the reaction. 4. Digest away plasmid DNA by incubation at 37 C for 30 min. 5. Terminate DNase reaction by addition of 200 L of TE. 6. Precipitate RNA by addition of 20 mL of 4...

Use of Flow Cytometry and Combined DNA Surface Staining for Analysis of Hematopoietic Development in the Xenopus Embryo

Incisional Healing Stages Pictures

Xenopus embryos provide a model for studying the earliest stages in the development of the vertebrate hematopoietic system. This chapter provides detailed procedures describing the production of hematopoietic chimeras in Xenopus embryos and the analysis of these chimeras using flow cytometry. Protocols for analysis include the determination of deoxyribonucleic acid content of hematopoietic cells, staining of cells with antibodies against cell surface antigens and the combined analysis of...

Cell Cycle Analysis of Embryonal Hematopoietic Cells

HSCs can be discriminated according to their position in the cell cycle. They are normally quiescent G0 G1 and are not high in metabolic activity. It is possible to quiescent discriminate cells by using different dyes. For example, Hoechst 33342 dye allows the separation of cells in G0 G1 from the cells in S G2-M. When a cell suspension stained with this dye is examined under two distinct wave lengths, a side population SP , characterized by weak fluorescence, can be identified and sorted 53,54...

Analysis of Hematopoietic Progenitors in the Mouse Embryo James Palis and Anne Koniski

Cell Palis

All mature blood cells are derived from hematopoietic progenitors that have been defined by their ability to generate colonies of cells in semisolid media. Investigation of the cellular components of these colonies has confirmed the existence of unilineage, bilineage, and multilineage progenitors. Furthermore, it has led to a better understanding of the relationships that exist among the erythroid, myeloid, and megakaryocyte lineages that compose much of the hematopoietic hierarchy in the...

Hemocyte Development During Drosophila Embryogenesis Robert A Schulz and Nancy Fossett

The model genetic organism Drosophila melanogaster has a rudimentary hematopoietic system with two embryonic blood cell types, crystal cells, and plasmatocytes. These distinct lineages provide the animal with an innate immune response and a means to remove apoptotic cells. Genetic analyses of Drosophila hematopoiesis have identified specific genes that function in blood cell formation. Complimentary deoxyribonucleic acid and antibody probes for these hematopoietic factors serve as important...

Imaging Early Macrophage Differentiation Migration and Behaviors in Live Zebrafish Embryos

Philippe Herbomel and Jean-Pierre Levraud Because zebrafish embryos are transparent, cell behaviors and interactions can be directly imaged noninvasely in live embryos using differential interference contrast-Nomarski light microscopy. We found that the imaging quality can be much improved by coupling differential interference contrast-Nomarski to true analogical color video so as to visualize the image in real time on a high-resolution colour video monitor. We explain here how to apply this...

Analysis of Hematopoietic Development in the Zebrafish Nolle N Paffett Lugassy and Leonard I

The zebrafish Danio rerio has emerged as a powerful vertebrate genetic and developmental model that is particularly amenable to the study of hematopoiesis. The zebrafish embryo develops externally and its optical clarity allows the number and morphology of circulating blood cells to be visualized using a dissecting microscope. Both the morphology of the blood lineages and the expression of critical blood genes are highly conserved between zebrafish and mammals. The high fecundity and short...

Pfhmii

Transmitted Light Dissecting Microscope

Ascorbic acid 5 mg mL 50 L a-MTG 26 L 2 mL IMDM 30-40 L 1X IMDM Pen-strep to 10 mL total 5 2 mM 25 g mL 0.45-0.6 mM 4 U mL 1X 2.3.6.3. Definitive Hematopoietic Colony Assays 1. Methocult , Stem Cells Technologies cat. no. M3434. Contains Epo and other cytokines. 2. 16-gage blunt-end needles, Stem Cells Technologies cat. no. HCC-8110. 3. Petri dishes, 3.5 cm, Becton Dickinson Falcon cat. no. 35-1008. 4. Tissue culture plates, 15 cm, Becton-Dickinson Falcon cat. no. 351013. 5. Preparation of...

Phenol Red Free Medium and Glass Bottom Vessels Improve Image Quality

The second parameter that contributes to image quality is background fluorescence. To test the influence of phenol red in the culture medium on background fluorescence, we acquired a series of images in media with and without this pH indicator. As shown in Fig. 3, phenol red dramatically increases the background levels, especially when visualizing GFP and RFP with the Endow GFP and TRITC filters, respectively. As a result, the relative signal intensity is decreased, hampering the detection of...

Notes

The authors intend to use this section to draw the reader's attention to several additional features of the StroCDB, in particular, the pages of the database that have not been discussed yet. These pages provide background information, additional methods, supplemental biological data, and interactive tables. 1. The Introduction button on the home page of StroCDB will retrieve http stromalcell. princeton.edu files intro.html, a page that provides a highly interactive perspective on stem cell...

Mouse Embryonic Explant Culture System for Analysis of Hematopoietic and Vascular Development

Embryo Scissor Tools

In vertebrates, the earliest differentiated cell types hematopoietic and endothelial arise from mesoderm induced during the process of gastrulation. These cells become organized into the blood islands of the extraembryonic yolk sac and are morphologically apparent by around d 7.5 in the mouse. Additional waves of hematopoietic and vasculogenic angio-genic activity later result in the development of definitive hematopoietic lineages and in the formation of the allantois and cardiovascular system...

Conditional Knockin

Lox Cre Knockin

The Cre-loxP strategy may also be applied for conditional expression of genes in a transgenic or knockin model Fig. 1A . A stopper fragment flanked by loxP Fig. 2. Engineering a conditionally targeted locus. A conditional gene-targeting construct contains a positive selection marker for selection of homologous recombinants flanked by two loxP sites and two homologous arms for directing homologous recombination into wild type locus A third loxP site is placed flanking a critical region of the...

Visualizing Individual Fluorescent Proteins With Different Filters

To compare and quantify the relative signal intensities of the different fluorescent proteins, we exposed individual cells expressing CFP, GFP, YFP, or RFP to light of different wavelengths, using the seven filters, for different lengths of time. As can be seen in Fig. 6, CFP top left can be best visualized by the Cyan GFP filter but also gives a signal with the Endow GFP and the JP1 filter. Thus, the Endow GFP filter Fig. 6. Fluorescent intensities of three GFP variants and of RFP determined...