Biomedical subjects

Margaret H Baron

Publications and source records attributed to Margaret H Baron.

12 recordsLinked to original sources

PubMed2006

Maturation and enucleation of primitive erythroblasts during mouse embryogenesis is accompanied by changes in cell-surface antigen expression.

Primitive erythroblasts (EryPs) are the first hematopoietic cell type to form during mammalian embryogenesis and emerge within the blood islands of the yolk sac. Large, nucleated EryPs begin to circulate around midgestation, when connections between yolk sac and embryonic vasculature mature. Two to 3 days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryPs in the circulation. The development and maturation of EryPs remain poorly defined. Our analysis of embryonic blood at different stages reveals a stepwise developmental progression within the EryP lineage from E9.5 to E12.5. Thereafter, EryDs are also present in the bloodstream, and the 2 lineages are not easily distinguished. We have generated a transgenic mouse line in which the human epsilon-globin gene promoter drives expression of green fluorescent protein exclusively within the EryP lineage. Here, we have used this line to characterize changes in cell morphology and surface-marker expression as EryPs mature and to track EryP numbers and enucleation throughout gestation. This study identifies previously unrecognized synchronous developmental stages leading to the maturation of EryPs in the mouse embryo. Unexpectedly, we find that EryPs are a stable cell population that persists through the end of gestation.

Animals↗

PubMed2006

Acceleration of mesoderm development and expansion of hematopoietic progenitors in differentiating ES cells by the mouse Mix-like homeodomain transcription factor.

The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mix-like (mMix) in gastrulation, its function in mesoderm development remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem cells in which mMix was genetically inactivated. Here we show that conditional induction of mMix in embryonic stem cell-derived embryoid bodies results in the early activation of mesodermal markers prior to expression of Brachyury/T and acceleration of the mesodermal developmental program. Strikingly, increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. Differentiation to primitive (embryonic) and definitive (adult type) blood cells proceeds normally and without an apparent bias in the representation of different hematopoietic cell fates. Therefore, the mouse Mix gene functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages.

Animals↗

PubMed2006

Tg(Afp-GFP) expression marks primitive and definitive endoderm lineages during mouse development.

Alpha-fetoprotein (Afp) is the most abundant serum protein in the developing embryo. It is secreted by the visceral endoderm, its derivative yolk sac endoderm, fetal liver hepatocytes, and the developing gut epithelium. The abundance of this protein suggested that Afp gene regulatory elements might serve to effectively drive reporter gene expression in developing endodermal tissues. To this end, we generated transgenic mouse lines Tg(Afp-GFP) using an Afp promoter/enhancer to drive expression of green fluorescent protein (GFP). Bright GFP fluorescence allowed the visualization, in real time, of visceral endoderm, yolk sac endoderm, fetal liver hepatocytes, and the epithelium of the gut and pancreas. Comparison of the localization of green fluorescence with that of endogenous Afp transcripts and protein indicated that the regulatory elements used to generate these mouse lines directed transgene expression in what appeared to be all Afp-expressing cells of the embryo, but only in a subset of fetal liver cells. The bright GFP signal permitted flow cytometric analysis of fetal liver hepatocytes. These mice represent a valuable resource for live imaging as well as identification, quantitation, and isolation of cells from the primitive and definitive endoderm lineages of the developing mouse embryo.

Animals↗

PubMed2005

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice.

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.

Aging↗

PubMed2005

Early patterning of the mouse embryo: implications for hematopoietic commitment and differentiation.

Prior to and during gastrulation, reciprocal interactions between embryonic and extraembryonic lineages are crucial for the correct patterning of the embryo. Several lines of investigation have underscored the importance of extraembryonic ectoderm and primitive endodermal in establishing the anterior-posterior axis of the embryo. Signals from these tissues help to position the primitive streak, from which mesoderm will emerge, within the epiblast (embryo proper). Molecules secreted by the visceral endoderm are required for activation of hematopoietic and endothelial cell development, but the pathways involved and their target tissue (e.g., posterior epiblast versus extraembryonic mesoderm) remain obscure. Recent evidence suggests that commitment of mesodermal progenitors to the hematopoietic and endothelial lineages begins earlier than previously anticipated, within or shortly after these cells emerge from the primitive streak.

Animals↗

PubMed2005

The specification of early hematopoiesis in the mammal.

PURPOSE OF REVIEW: A number of interesting surprises has emerged during the past year in the field of early hematopoietic development. This review highlights recent studies that have challenged the prevailing view of embryonic and fetal hematopoiesis in mammals, with a focus on the mouse as a model system. The authors apologize to the many colleagues whose work could not be cited because of space limitations. RECENT FINDINGS: Advances in our understanding of the embryonic origins of hematopoiesis in mammals and in the regulation of primitive and definitive hematopoietic development are discussed. SUMMARY: The ontological relation between primitive (embryonic) and definitive (fetal and adult) hematopoiesis still holds some mysteries for the biologist. Both technical and conceptual breakthroughs have refined our view of how blood cells form at different stages of development. What we learn from the embryo is not only of fundamental interest but may have future applications in the clinic.

Animals↗

PubMed2005

Mouse embryonic explant culture system for analysis of hematopoietic and vascular development.

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/angiogenic activity later result in the development of definitive hematopoietic lineages and in the formation of the allantois and cardiovascular system of the embryo proper. In part because of the limited accessibility of the mammalian embryo to experimental manipulation in vivo, regulation of these events is still not well understood. Both in the yolk sac and within the embryo proper, prospective hematovascular mesoderm differentiates in the vicinity of endodermal tissues. Here we describe an embryonic explant culture system that permits the dissection of mesodermal and endodermal contributions to hematopoietic and endothelial cell formation during gastrulation. This system can be used to assay for soluble or endodermal cell-associated molecules involved in mediating critical interactions between mesoderm and endoderm in the formation of the hematopoietic and endothelial lineages during embryonic development.

Animals↗

PubMed2005

Dynamic in vivo imaging of mammalian hematovascular development using whole embryo culture.

The yolk sac is the initial site of hematopoiesis in the mammalian embryo. As the embryo develops, blood vessels form around primitive erythroblasts to connect the yolk sac to the embryo, delivering newly formed blood cells to the embryonic circulation. The limited accessibility of the mammalian embryo has made it difficult to study the dynamic changes in cellular development during the formation of the early hematovascular system. Therefore, we have developed a culture system for studying early hematopoiesis, vasculogenesis. and angiogenesis in the mouse embryo. Early embryos (E7.5-E9.5) can be grown on the microscope stage to study the dynamics as vessels form and circulation begins. In addition, this mouse embryo culture system provides an excellent model for understanding the interplay between flow dynamics and cellular development.

Animals↗

PubMed2004

Bone morphogenetic proteins in vertebrate hematopoietic development.

During embryonic development, the hematopoietic system is the first to generate terminally differentiated, functional cell types. The urgent necessity for the early formation of blood and blood vessels during embryogenesis means that the induction, expansion, and maturation of these systems must be rapidly and precisely controlled. Bone morphogenic proteins (BMPs) have been implicated in hematopoietic development in the vertebrate embryo and stimulate the proliferation and/or differentiation of human cord blood hematopoietic stem cells (HSC) and embryonic stem cells in vitro. Here we review the mechanisms of action and potential roles of these soluble signaling molecules in vertebrate hematopoiesis.

Animals↗

PubMed2003

Mouse Mix gene is activated early during differentiation of ES and F9 stem cells and induces endoderm in frog embryos.

In frog and zebrafish, the Mix/Bix family of paired type homeodomain proteins play key roles in specification and differentiation of mesendoderm. However, in mouse, only a single Mix gene (mMix) has been identified to date and its function is unknown. We have analyzed the expression of mouse Mix RNA and protein in embryos, embryoid bodies formed from embryonic stem cells and F9 teratocarcinoma cells, as well as several differentiated cell types. Expression in embryoid bodies in culture mirrors that in embryos, where Mix is transcribed transiently in primitive (visceral) endoderm (VE) and in nascent mesoderm. In F9 cells induced by retinoic acid to differentiate to VE, mMix is coordinately expressed with three other endodermal transcription factors, well before AFP, and its protein product is localized to the nucleus. In a subpopulation of nascent mesodermal cells from embryonic stem cell embryoid bodies, mMix is coexpressed with Brachyury. Intriguingly, mMix mRNA is detected in a population (T+Flk1+) of cells which may contain hemangioblasts, before the onset of hematopoiesis and activation of hematopoietic markers. In vitro and in vivo, mMix expression in nascent mesoderm is rapidly down-regulated and becomes undetectable in differentiated cell types. In the region of the developing gut, mMix expression is confined to the mesoderm of mid- and hindgut but is absent from definitive endoderm. Injection of mouse mMix RNA into early frog embryos results in axial truncation of developing tadpoles and, in animal cap assays, mMix alone is sufficient to activate expression of several endodermal (but not mesodermal) markers. Although these observations do not exclude a possible cell-autonomous function for mMix in mesendodermal progenitor cells, they do suggest an additional, non-cell autonomous role in nascent mesoderm in the formation and/or patterning of adjacent definitive endoderm.

Animals↗

PubMed2003

Embryonic origins of mammalian hematopoiesis.

Hematopoiesis and vasculogenesis in the mammalian embryo begin in the blood islands of the yolk sac and continue, somewhat later, within the embryo proper. A subset of the first endothelial and hematopoietic cells of the yolk sac arise in close spatial and temporal association, apparently from a common mesodermal progenitor, the "hemangioblast." The mechanisms that control formation of hemangioblast and embryonic hematopoietic and endothelial (angioblastic) stem/progenitor cells are still not well understood. Formation of these cell types from nascent mesoderm requires signals from an adjacent outer layer of primitive (visceral) endoderm. Indian hedgehog (Ihh), a member of the hedgehog family of extracellular morphogens, is secreted by visceral endoderm and alone is sufficient to induce hematopoiesis and vasculogenesis in explanted embryos. While gene targeting studies in mice support a role for hedgehog signaling in these processes in vivo, they also suggest that additional molecules (perhaps, for example, Wnt proteins) are required for induction and patterning of hematopoietic and vascular mesoderm. Indian hedgehog likely functions through upregulation of genes encoding other signaling molecules, such as bone morphogenetic protein (Bmp)-4, in the target tissue. This review will focus on hematopoietic and vascular development in the early mouse embryo and will discuss potential implications of recent studies for stem cell transplantation in humans.

Animals↗

PubMed2002

Structure, upstream promoter region, and functional domains of a mouse and human Mix paired-like homeobox gene.

Mix/Bix proteins represent a vertebrate subgroup of paired-like homeodomain proteins which are known to function around the time of gastrulation. Here we report the structures of the genomic and upstream promoter regions of a mouse and human Mix-like gene. Both genes map to syntenic regions of chromosome 1 and contain two coding exons, with the paired-type homeodomain split between the exons within helix 3. Differentiating mouse embryonic stem cells transcribe a messenger RNA of approximately 2.6 kb. The first exon encodes the translation initiation codon and a 5' untranslated region of approximately 90 bp. Sequence analysis of the 960 bp upstream of the transcription start site of the mouse Mix gene revealed the presence of a putative initiator region and TATA box as well as potential Smad, FoxH1/FAST, T-box, COUP-TF, C/EBP, GATA, HNF3 binding sites and retinoic acid response elements. A number of these sites are conserved in the human Mix promoter. We find that most paired-related homeodomain proteins, including mouse and human Mix, contain a proline-rich region within their amino termini which may interact with other proteins. Mouse and human Mix proteins contain highly conserved carboxy-terminal polar/acidic regions with the potential to form an amphipathic helix and the ability to activate transcription in yeast. Mouse Mix expressed in COS cells or in vitro binds a DNA consensus sequence identified previously for paired class homeodomain proteins. These studies suggest that a number of features of paired-like protein structure and function are conserved across diverse species and provide a useful framework for studying the function and regulation of the mouse Mix gene.

Amino Acid Sequence↗