ABSTRACT
The relative contribution of yolk sac and intraembryonic precursors to hematopoiesis has been a matter of long-standing controversy. As reconstitution activity has so far only been found in embryonic tissues after the onset of circulation, the origin of reconstituting cells could not be formally established. Here, we separated yolk sac and intraembryonic splanchnopleura prior to circulation and maintained the explants in organ culture before transfer. Precursors derived from the intraembryonic site generated multilineage hematopoietic progeny in adult mice for more than 6 months. Yolk sac cells only provided myeloid short-term reconstitution. The results reveal a differential hematopoietic capacity of precirculation embryonic tissues in vivo, and indicate that the only cells capable of adult long-term hematopoiesis are of intraembryonic origin.
Subject(s)
Embryo, Mammalian/cytology , Hematopoietic Stem Cells/physiology , Yolk Sac/cytology , Animals , Cell Differentiation , Cell Lineage , DNA-Binding Proteins , Female , Histocompatibility Antigens Class I/analysis , Mice , Mice, Inbred C57BL , Organ Culture Techniques , PregnancyABSTRACT
We have identified and characterized the stem cell antigen AA4. This molecule is a type I transmembrane protein whose overall structure suggests a role in cell adhesion. During fetal ontogeny (days 9-14 of development), AA4 is expressed in three major cell types: vascular endothelial cells, aorta-associated hematopoietic clusters, and primitive fetal liver hematopoietic progenitors. In the adult, AA4 is abundant in lung, heart, and whole bone marrow. In the adult hematopoietic compartment, aa4 transcripts are present in bone marrow CD34(-/lo) Lin- Sca-1+ c-Kit+ and CD34hi Lin- Sca-1+ c-Kit+ stem and progenitor cell subsets. Our observations suggest that AA4 plays a role in cell-cell interactions during hematopoietic and vascular development.
Subject(s)
Antigens, Surface/chemistry , Fetus/immunology , Hematopoietic Stem Cells/immunology , 3T3 Cells , Amino Acid Sequence , Animals , Antigens, Surface/biosynthesis , Base Sequence , Biomarkers/chemistry , Cloning, Molecular , Hematopoietic Stem Cells/chemistry , Hematopoietic Stem Cells/metabolism , Humans , In Situ Hybridization , Mice , Mice, Inbred C57BL , Molecular Sequence Data , RNA, Messenger/analysis , Recombinant Proteins/biosynthesis , Sequence Alignment , Sequence Analysis, DNAABSTRACT
It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, we analysed the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc), with a particular emphasis on intraembryonic hemogenic sites. We show here that both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development. (1) Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase of intraembryonic precursors determination. (2) A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved in the specification of intraembryonic hemopoietic precursors. (3) Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during fetal liver colonisation. Furthermore, a strong GATA-3 signal allowed us to uncover previously unreported mesodermal aggregates beneath the aorta. A combined in situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation.
Subject(s)
DNA-Binding Proteins/genetics , Embryonic and Fetal Development , Gene Expression Regulation, Developmental , Hematopoietic Stem Cells/physiology , Metalloproteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Adaptor Proteins, Signal Transducing , Allantois/cytology , Allantois/physiology , Animals , Aorta/embryology , Brain/embryology , GATA3 Transcription Factor , Gastrula/cytology , Gastrula/physiology , Germ Cells/physiology , Hematopoietic Stem Cells/cytology , LIM Domain Proteins , Mesoderm/physiology , Mice , Mice, Inbred BALB C , Transcription, GeneticABSTRACT
The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. SCL is also expressed in the central nervous system, although its expression pattern has not been examined in detail and its function in neural development is unknown. In this article we present the first analysis of SCL transcriptional regulation in vivo. We have identified three spatially distinct regulatory modules, each of which was both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain, namely, developing endothelium, midbrain, and hindbrain/spinal cord. In addition we have demonstrated that GATA factor binding sites are essential for neural expression of the SCL constructs. The midbrain element was particularly powerful and axonal lacZ expression revealed the details of axonal projections, thus implicating SCL in the development of occulomotor, pupillary, or retinotectal pathways. The neural expression pattern of the SCL gene was highly conserved in mouse, chicken, and zebrafish embryos and the 5' region of the chicken SCL locus exhibited a striking degree of functional conservation in transgenic mice. These data suggest that SCL performs critical functions in neural development. The regulatory elements identified here provide important tools for analyzing these functions.