Thierry Jaffredo

From mesoderm to blood islands: patterns of key molecules during yolk sac erythropoiesis.

Several identified genes play key roles in the specification of the blood-forming system, from commitment of mesoderm to differentiation of hemopoietic and endothelial cells. We have thoroughly analyzed the expression dynamics of some of these genes during yolk sac erythropoiesis in the chick embryo. The study includes transcription factors which are known to participate in multimeric complexes: GATA-1, -2, SCL/tal-1 and Lmo2 (whose avian orthologue we have cloned), VEGF-R2, a critical regulator of hemopoietic and endothelial commitment, and hemoglobin used as a marker of the last step in erythroid differentiation. Several findings were unexpected. (1) Two distinct patterns were revealed for GATA-2, first: low expression, ubiquitous in all mesodermal cells, as soon as cells ingress through the primitive streak; secondly: high, blood island-specific expression. (2) VEGF-R2 is coexpressed with GATA-2 at the level of the primitive streak. (3) SCL and Lmo2 expression is restricted to presumptive hemangioblasts. (4) The up-regulation of GATA-2 in newly formed blood islands is shortly followed by GATA-1 expression. (5) Lmo2 is up-regulated in blood island angioblasts thus appearing as one of the earliest markers for endothelial cell commitment. VEGF-R2 is down-regulated in hemopoietic cells prior to GATA-2, SCL/tal-1, Lmo2 and GATA-1 in erythroblasts.

MC29-immortalized clonal avian heart cell lines can partially differentiate in vitro

We established quail clonal heart muscle cell lines from cardiac rhabdomyosarcomas developed in embryos injected in ovo with the MC29 virus containing the v-myc oncogene. These clones were characterized by means of antibodies detecting markers of striated muscle cells. Two clones were selected for further characterization on the basis of a distribution of myogenic markers similar to that in normal early embryonic cardiac muscle cells. However, these muscle markers progressively disappeared with time in culture. Cardiomyocytic differentiation could be reinduced in culture, by associating the avain cardiac cells with 3T3 cells in a defined synthetic medium. Muscle markers were then reexpressed in all cardiac cells as soon as Day 1 after coculture. Multiplication of cardiac cells continued at the same time. This is characteristic of cardiac clones since MC29-infected quail myoblasts and MC29-infected quail fibroblasts exhibited a split response to 3T3 association, i.e., decreased growth and enhanced differentiation. The cardiac clones were maintained in vitro for more than 60 generations (6 months) without morphological changes. To our knowledge, this is the first description of clonal embryonic avian heart cell lines.

In vivo diversification and migrations of chick embryo heart muscle cells: a morphometric analysis with ALV- and SNV-based non-replicative vectors.

Abstract By means of a reporter gene we previously demonstrated that non-replicative Avian Leukemia Virus- and Spleen Necrosis Virus-based retroviral vectors were preferentially expressed in the heart of avian embryos from different species. Using a computer-assisted approach, we now compare clones tagged by the two types of vectors, for volume, anatomical and subanatomical localisation, number of Hoechst-stained cell nuclei and mean cell division time during the period of heart morphogenesis, i.e. from stages 17–19 to 34 of Hamburger and Hamilton (1951). This analysis demonstrates that clones labelled by the two types of viruses display similar features and bring about new insights on the relationships between mitotic and migratory properties of the myocardial cells and histogenesis of the heart. Since only exteriormost cells were tagged with our inoculation procedure, our analysis shows that: (1) at stages 17–19, the myocardium is composed of cells with diverse potentials; some cells still retain the capacity to divide extensively and participate to different heart muscle layers, whilst most are restricted in their multiplication potential and contribute to single muscle layers; (2) about half of the clones are located deep in the heart wall, revealing extensive cell migrations from the heart surface to the ventricular trabeculae, the first migrating cells tagged being detected 20 h after viral inoculation. The presence of these cells is consistent with the finding of a large number of compact trabecular clones 5 days later suggesting that these cells divide mainly after completing migration. Our approach provides new insights as well as quantitative data on the different processes involved in heart morphogenesis, namely multiplication, migration and localisation of heart muscle cells.

Intra-Aortic Hematopoietic Cells

Life-long function of the blood-forming system depends on a pool of self-renewable Hematopoietic Stem Cells (HSCs). During ontogeny, these cells seed the rudiments of hematopoietic and lymphoid organs, whether they are mesodermal (bone marrow, spleen, milky spots of the omentum, secondary lymphoid organs) or endodermal/mesodermal (thymus, bursa of Fabricius, fetal liver). The only exception is the yolk sac, which produces its own progenitors and stem cells. To grasp how the adult HSCs pool is maintained, it is important to understand how HSCs become committed and segregated during development. It was once thought that these cells emerged once for all, early in ontogeny, in the yolk sac (or, in amphibians, in the yolk sac-equivalent, the ventral blood island) [1]. However it was known that the cellular and molecular features of blood cells, notably red cells, changed along the course of development, a fact that might indicate either an environmental influence of the differentiation site or intrinsic properties of successive generations of HSCs. An experimental model, consisting of a quail embryo developing on a chicken yolk sac, then disclosed the existence of an intra-embryonic origin source of HSCs [2]. In these chimeras the definitive hematopoietic organs were colonized by HSCs from the embryo and hemoglobin switches could be related to the emergence of these intra-embryonic HSCs [3].