Core binding factor in the early avian embryo: cloning of Cbfbeta and combinatorial expression patterns with Runx1.

We have isolated the avian ortholog for CBFbeta, the common non-DNA binding subunit of the core binding factor (CBF) that has important regulatory roles in major developmental pathways. CBFbeta forms heterodimers with the DNA-binding Runx proteins and increases their affinity for DNA and their protein stability. Here, we describe the Cbfbeta expression pattern during the first 4 days of chick embryo development, with a special interest in the developing hematopoietic system. We have compared its expression pattern to that of Runx1, which is crucial for the generation of definitive hematopoietic cells, and to other hematopoietic- or endothelial-specific markers (c-Myb, Pu.1, CD45, c-Ets-1 and VE-Cadherin). Initially, Cbfbeta is widely expressed in the early mesoderm in both the yolk sac and the embryo proper, but later its expression becomes restricted to specific organs or cell types. We have found that Cbfbeta expression overlaps with Runx1 in the hematopoietic system and neural tube. The somitic and mesonephric structures, however, express Cbfbeta in the absence of detectable Runx1. Finally, Cbfbeta and Runx1 display multiple combinatorial patterns in the endoderm and in specific nerves or ganglia. Taken together, we show that Cbfbeta exhibits a dynamic expression pattern that varies according to the organ, cell type or developmental stage. By revealing multiple combinatorial patterns between Cbfbeta and Runx1, these data provide new insights into the role of CBF during early development.

Molecular cloning and embryonic expression of the Xenopus Arnt gene.

In this paper, we report the cloning of a Xenopus bHLH/PAS factor homologous to the mammalian aryl hydrocarbon receptor nuclear translocator (Arnt) or Drosophila Tango gene. Sequence data analysis indicates that protein domains organization in xArnt is strongly conserved and that xArnt is highly related to the mammalian Arnt1 isoform. As revealed by reverse transcriptase polymerase chain reaction and whole-mount in situ hybridization, xArnt gene is expressed during early and late development. At early stages, xArnt transcripts are restricted to the ectoderm and extends to the marginal zone at gastrula stage. In tail bud embryo, xArnt is strongly expressed in branchial arches, optical and optical vesicles, and pronephros and pronephritic duct.

Characterization and developmental expression of xSim, a Xenopus bHLH/PAS gene related to the Drosophila neurogenic master gene single-minded.

We have isolated a novel gene from Xenopus, denominated xSim, which encodes a protein of 760 amino acids containing a basic helix-loop-helix (bHLH) motif contiguous to a PAS domain characteristic of an emerging family of transcriptional regulators so called bHLH/PAS. xSim shares a strong amino acid sequence identity with the Drosophila Single-minded (dSim) and with the murine Sim1 and Sim2 proteins. Phylogenetic analysis reveals that xSim gene is an ortholog gene of the mSim2 gene. Spatio-temporal analysis shows a maternal and a zygotic expression of xSim throughout early Xenopus development. In situ hybridization assays reveal that the transcripts are enriched in the animal hemisphere until blastula stage and extend to the marginal zone at early gastrula stage. As development proceeds, xSim is mainly restricted to the central nervous system.

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.