Margaret H. Baron

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta–gonad–mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, β-NGF (a neurotrophic factor), MIP-1γ (a C–C chemokine family member) and Bmp4 (a TGF-β family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

Winged-Helix, Hedgehog and Bmp genes are differentially expressed in distinct cell layers of the murine yolk sac

The visceral yolk sac plays a critical role in normal embryogenesis, yet little is known about the specific molecules that regulate its development. We show here that four winged-helix genes (HNF-3alpha, HNF-3beta, HNF-3gamma and HFH-4) are restricted to visceral endoderm. In the absence of HNF-3beta, visceral endoderm forms but the morphogenetic movements by which the embryo becomes enclosed within its yolk sac are disrupted and serum protein gene transcription is greatly reduced. Hedgehog and Bmp genes, which encode signaling molecules known to play multiple roles in embryonic development, are also differentially expressed in the closely apposed yolk sac mesoderm and endoderm layers. Our results suggest that similar mechanisms may be utilized to mediate inductive interactions in both extraembryonic and embryonic tissues.

Antibodies against the chemically synthesized genome-linked protein of poliovirus react with native virus-specific proteins

The genome-linked protein (VPg) of poliovirus has been chemically synthesized, coupled to bovine serum albumin carrier and injected into rabbits. An antibody response was elicited not only by the full-length synthetic VPg peptide, but also by a synthetic 14-amino acid carboxy-terminal peptide. All antisera reacted with virus-specific proteins from HeLa cells infected with poliovirus. Three of these proteins have previously been implicated by others as precursors of VPg. No free cytoplasmic VPg could be detected, and the antibodies did not react with radiolabeled proteins from uninfected cells.

The embryonic origins of erythropoiesis in mammals

Erythroid (red blood) cells are the first cell type to be specified in the postimplantation mammalian embryo and serve highly specialized, essential functions throughout gestation and postnatal life. The existence of 2 developmentally and morphologically distinct erythroid lineages, primitive (embryonic) and definitive (adult), was described for the mammalian embryo more than a century ago. Cells of the primitive erythroid lineage support the transition from rapidly growing embryo to fetus, whereas definitive erythrocytes function during the transition from fetal life to birth and continue to be crucial for a variety of normal physiologic processes. Over the past few years, it has become apparent that the ontogeny and maturation of these lineages are more complex than previously appreciated. In this review, we highlight some common and distinguishing features of the red blood cell lineages and summarize advances in our understanding of how these cells develop and differentiate throughout mammalian ontogeny.

The fetal liver is a niche for maturation of primitive erythroid cells

Primitive erythroid cells (EryP) are the earliest differentiated cell type of the mammalian embryo. They appear in the yolk sac by embryonic day 7.5, begin to enter the embryonic circulation 2 days later and continue to mature in a stepwise and synchronous fashion. Like their adult counterparts, EryP enucleate. However, EryP circulate throughout the embryo for several days before the first enucleated forms can be identified in the blood. We have used transgenic mouse lines in which GFP marks EryP to investigate this seemingly long lag and have identified a previously unrecognized developmental niche for EryP maturation. After exiting the yolk sac, EryP begin to express cell adhesion proteins, including α4, α5, and β1 integrins, on their surface and migrate into the fetal liver (FL), where they interact with macrophages within erythroblastic islands. Binding of EryP to FL macrophages in vitro is stage-specific and partly depends on VCAM-1. The ability to tag and track EryP nuclei using a transgenic mouse line expressing an H2B-EGFP fusion allowed us to identify and characterize extruded EryP nuclei and to demonstrate that molecules such as α4, α5, and β1 integrins are redistributed onto the plasma membrane surrounding the extruding nucleus. FL macrophages engulf extruded EryP nuclei in cocultures and in the native FL in vivo. We conclude that EryP home to, complete their maturation, and enucleate within the FL, a tissue that is just developing as EryP begin to circulate. Our observations suggest a simple solution for a puzzling aspect of the development of the primitive erythroid lineage.

A membrane associated mCherry fluorescent reporter line for studying vascular remodeling and cardiac function during murine embryonic development

The development of the cardiovascular system is a highly dynamic process dependent on multiple signaling pathways regulating proliferation, differentiation, migration, cell–cell and cell‐matrix interactions. To characterize cell and tissue dynamics during the formation of the cardiovascular system in mice, we generated a novel transgenic mouse line, Tg(Flk1::myr‐mCherry), in which endothelial cell membranes are brightly labeled with mCherry, a red fluorescent protein. Tg(Flk1::myr‐mCherry) mice are viable, fertile, and do not exhibit any developmental abnormalities. High levels of mCherry are expressed in the embryonic endothelium and endocardium, and expression is also observed in capillaries in adult animals. Targeting of the fluorescent protein to the cell membrane allows for subcellular imaging and cell tracking. By acquiring confocal time lapses of live embryos cultured on the microscope stage, we demonstrate that the newly generated transgenic model beautifully highlights the sprouting behaviors of endothelial cells during vascular plexus formation. We have also used embryos from this line to imaging the endocardium in the beating embryonic mouse heart, showing that Tg(Flk1::myr‐mCherry) mice are suitable for the characterization of cardio dynamics. Furthermore, when combined with the previously described Tg(Flk1::H2B‐EYFP) line, cell number in addition to cell architecture is revealed, making it possible to determine how individual endothelial cells contribute to the structure of the vessel. Anat Rec, 2008.

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. Developmental Dynamics 235:2549–2558, 2006.

Cooperative Transcriptional Activation by Serum Response Factor and the High Mobility Group Protein SSRP1

Serum response factor (SRF) is a MADS box transcription factor that controls a wide range of genes involved in cell proliferation and differentiation. The MADS box mediates homodimerization and binding of SRF to the consensus sequence CC(A/T)6GG, known as a CArG box, which is found in the control regions of numerous serum-inducible and muscle-specific genes. Using a modified yeast one-hybrid screen to identify potential SRF cofactors, we found that SRF interacts with the high mobility group factor SSRP1 (structure-specific recognition protein). This interaction, which occurs in yeast and mammalian cells, is mediated through the MADS box of SRF and a basic region of SSRP1 encompassing amino acids 489–542, immediately adjacent to the high mobility group domain. SSRP1 does not bind the CArG box, but interaction of SSRP1 with SRF dramatically increases the DNA binding activity of SRF, resulting in synergistic transcriptional activation of native and artificial SRF-dependent promoters. These results reveal an important role for SSRP1 as a coregulator of SRF-dependent transcription in mammalian cells.

Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo

Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-β1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation.

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. Developmental Dynamics 226:000–000, 2003.