Developmental hematopoiesis is an exciting multi-disciplinary field at the crossroads of developmental biology, hematopoiesis, stem cell biology and genetics. This field began nearly a century ago with detailed observations on the formation of the first blood cells, the primitive erythrocytes, in the yolk sac of the developing chicken embryo, and it has incorporated many new scientific techniques and adopted alternative model organisms throughout its history. More recently, the intense focus on the clinical potential of stem cells has underscored the importance of understanding the developmental origins of the hematopoietic system and has encouraged research in this area. In this rapidly moving field, Developmental Hematopoiesis: Methods and Protocols is a timely book that provides a comprehensive overview of current methods and protocols available in developmental hematopoiesis. When I first saw this book advertised, I immediately ordered a copy for my lab. Yes,I do have a book fetish, but how could a group working in the field of developmental hematopoiesis do without a volume with such a promising title? I can now attest that this book is indeed worth its purchase price.
The editor, Margaret Baron, has put together a list of chapters covering an impressive breadth of topics contributed by recognized experts in developmental hematology. All the chapters start with a brief overview of their subject with references to the relevant literature or to other protocol books where appropriate; this is especially helpful to novices in the field. Most chapters contain in-depth coverage of the materials, protocols and potential pitfalls of the technique under discussion, while nuggets of wisdom are found in the `Notes' section at the end of each chapter. The latter are extra bits of valuable information of the type that usually do not make it into journal articles, and their inclusion in this volume gives the reader a sense of being taught on a one-to-one basis. For example, it is useful to be cautioned against injecting zebrafish embryos through the yolk as this is sticky and will clog the needle, and to be warned not to attempt to isolate mouse epiblast and visceral endoderm from more than four litters at once to avoid having explants sit around for too long before culture.
The book is divided into four sections. The first section provides an overview of the genetic approaches that are available in the mouse model for studying the molecular mechanisms of developmental hematopoiesis. The second part deals with mouse transplantation models and their analyses. Experimental assays in various model organisms ranging from Drosophila to human,rightfully take up the largest part of the book (327 out of the 480 pages). This allocation probably reflects the growing realization that the overall molecular mechanisms that drive hematopoiesis in ontogeny are well conserved between species, and that important insights can perhaps be more readily obtained from non-mammalian model systems. The last section provides an in-depth discussion of recent functional genomic approaches to the study of hematopoietic stem cell biology, with a special focus on the stem cell niche. Together, the chapters in these sections provide a good overview of the experimental approaches available in the different model organisms. Although there is some unavoidable overlap between chapters, this is not a problem because it more frequently results in the conveyance of additional information than in exact duplication.
It is not surprising that the majority of the experimental protocols presented in this book relate to the mouse model, as this is the most widely used system in which to study developmental hematopoiesis. The strengths of the mouse model (apart from it being mammalian) include the availability of antibodies for the identification and purification of different classes of hematopoietic stem, progenitor and mature blood cells; the variety of in vitro and in vivo functional assays; the availability of genetics and genomics; and the existence of in vitro embryonic stem (ES) cell culture systems that recapitulate the earliest events in embryonic blood cell formation. All these aspects and more are addressed in the current volume. There is an overview of knockout, knock-in and transgenic strategies in mice, from which one can choose the best approach to study the hematopoietic role of a gene of interest. Another chapter explains the complex mouse breeding strategies required to identify quantitative trait loci involved in hematopoietic stem cell (HSC) biology. Several chapters comprehensively address the isolation and manipulation of (pre-) hematopoietic tissues, including the epiblast, yolk sac, the para-aortic splanchnopleura/aortagonad-mesonephros region, liver and thymus, from different developmental stages of mouse embryos for functional analysis. These tissues, or HSCs purified from them, can be assayed for their hematopoietic potential in vitro in, for example, hematopoietic induction assays (epiblast), colony-forming assays in culture, whole-explant cultures on stromal cell lines that support hematopoiesis, fetal thymus organ cultures and a derivative thereof that allows for the analysis of the lineage potential of single cells, and in vivo in transplantation assays to monitor HSC potential in hematopoietic and vascular lineages. The common techniques for adoptive transfer into adult recipients are not addressed in detail in this book, as these protocols are well established and described in detail in a previous volume of this series. This book does, however, offer instructive protocols for the lesser known, but equally important, neonatal and in utero transplantation strategies. For those preferring cell models over embryos,there are detailed protocols for the hematopoietic and vascular differentiation of mouse ES cells, and for the inducible expression of transgenes in ES cells. Methods are also presented for the in vitro expansion of primary multipotent and erythroid progenitors from mouse fetal liver or human cord blood for biochemical and other analyses that require larger cell numbers. Finally, two detailed chapters on imaging describe a whole-mouse-embryo culture system for studying the onset of hematopoiesis in the yolk sac and a procedure to follow the fate of individual hematopoietic cells in vitro, including information on the optimal filters to use for detecting GFP, CFP, YFP and RFP, singly and multiply.
The human system is represented in two chapters: one on the analysis of hematopoietic sites and cells in the human embryo; and one on the generation of hematopoietic cells from human ES cells, which may have important implications for future therapies. For most investigators, the use of early human embryos will not be a possibility, but the detailed description is helpful in highlighting the differences between the human and the mouse model,and in appreciating the difficulties in human embryo research.
The non-mammalian model organisms covered in the book are the chicken, Xenopus, zebrafish and the fruit fly Drosphila melanogaster. The chicken embryo has a long-standing history in developmental hematopoiesis. Seminal experiments on the developmental origins of the hematopoietic system were performed in this species, as its accessibility and flat morphology make it well suited for the grafting experiments described in Developmental Hematopoiesis.
Xenopus, zebrafish and Drosophila all produce large numbers of offspring that develop externally. The unique feature that made Xenopus a valuable model organism for developmental hematopoiesis is the clear spatial separation of primitive and definitive hematopoiesis, which takes place in the ventral blood islands and dorsal lateral plate mesoderm,respectively. This spatial separation allows for the detailed analysis of the developmental origin of these two distinct cell populations. Two alternative approaches to map the hematopoietic fate of pre-gastrulation embryonic regions– and how to interfere with this – are discussed in detail. In addition, similar to the chicken, the externally development of the embryos makes it possible to perform grafting experiments to examine the hematopoietic potential of various mesodermal tissues, and detailed protocols for this type of experiments are provided.
The power of genetics in zebrafish and Drosophila make these the model organisms of choice in, for example, high-throughput genetic screening. Among the strengths of the zebrafish model are the relatively short generation time, the transparency and accessibility of its embryos, and the presence of a`complete' hematopoietic system with myeloid and lymphoid cell lineages. Two excellent chapters describe the basic techniques for the manipulation and analysis of hematopoiesis in the zebrafish embryo by in situ hybridization,(transient) transgenesis, morpholino-mediated knock down of genes of interest and imaging of live embryos.
Although the hematopoietic system in Drosophila is limited to myeloid-like cell lineages, studies into the embryonic development of these cells have revealed a remarkable conservation of the molecular players required at the onset of hematopoiesis between Drosophila and vertebrate species. The detailed chapter on how to analyze hemocyte development by in situ hybridization and immunohistochemistry will be helpful to those of us who wish to make use of fruit fly genetics to identify novel players in developmental hematopoiesis.
Overall, Developmental Hematopoiesis: Methods and Protocols more than meets its aim to be a resource for students, post-docs and more experienced investigators interested in the origins of hematopoiesis. Although personal training might still be required to perform the more-complex experiments successfully, such as dissections of early embryos, in utero transplantation or injection of zebrafish/Xenopus embryos, the wealth of detail and information in the individual chapters make this book a valuable guide to our understanding and use of the available experimental strategies in this growing field.