Identification of Novel Hemangioblast Genes in the Early Chick Embryo

Abstract

During early vertebrate embryogenesis, both hematopoietic and endothelial lineages derive from a common progenitor known as the hemangioblast. Hemangioblasts derive from mesodermal cells that migrate from the posterior primitive streak into the extraembryonic yolk sac. In addition to primitive hematopoietic cells, recent evidence revealed that yolk sac hemangioblasts also give rise to tissue-resident macrophages and to definitive hematopoietic stem/progenitor cells. In our previous work, we used a novel hemangioblast-specific reporter to isolate the population of chick yolk sac hemangioblasts and characterize its gene expression profile using microarrays. Here we report the microarray profile analysis and the identification of upregulated genes not yet described in hemangioblasts. These include the solute carrier transporters SLC15A1 and SCL32A1, the cytoskeletal protein RhoGap6, the serine protease CTSG, the transmembrane receptor MRC1, the transcription factors LHX8, CITED4 and PITX1, and the previously uncharacterized gene DIA1R. Expression analysis by in situ hybridization showed that chick DIA1R is expressed not only in yolk sac hemangioblasts but also in particular intraembryonic populations of hemogenic endothelial cells, suggesting a potential role in the hemangioblast-derived hemogenic lineage. Future research into the function of these newly identified genes may reveal novel important regulators of hemangioblast development.

Keywords: chicken embryo; hemangioblast; microarray analysis; novel genes; yolk sac.

Figure 1

Expression of the yolk sac hemangioblast reporter in the early chick embryo (HH5). (A) Chick embryo co-electroporated with the pCAGGS-RFP ubiquitous reporter (red) and the Hb-eGFP hemangioblast reporter (green); (B) Transverse section of an Hb-eGFP-electroporated embryo immunolabeled for cVEGFR2 (magenta). At this early stage, the Hb-eGFP reporter specifically labels the yolk sac population of hemangioblasts, which can be identified by the expression of cVEGFR2 (B). This membrane receptor is detected at the surface of the eGFP-expressing cells. BF, bright field. Scale bar: 100 µm.

Figure 2

Ingenuity pathway analysis of genes differentially expressed in yolk sac hemangioblasts. (A) Top classes of biological functions and (B) canonical signaling pathways most significantly represented in the Hb-eGFP+ microarray dataset (Ingenuity Pathways Analysis (IPA) library; Ingenuity Systems, www.ingenuity.com). Genes that met the fold change cutoff of 1.2 were considered for the analysis. Bars indicate the minus log of the p-value of each functional class/canonical pathway. The threshold line (orange) corresponds to a p-value of 0.05. The yellow line in (B) represents the ratio between the number of genes from the dataset in a given pathway that meet the cutoff criteria and the total number of genes of that pathway; (C) Network diagram representing the molecular relationships between genes differentially expressed in hemangioblasts. This graphical representation generated by IPA includes gene products of four functional classes: Cellular Development, Cardiovascular System Development and Function, Organismal Development, Organ Development and Cell Signaling. Gene products are represented as nodes (shapes) and the biological relationship between two nodes is represented as an edge (line). Orange lines represent interactions between gene products from different canonical pathways. All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base. The intensity of the node color indicates the degree of upregulation (red) or downregulation (green). Nodes are displayed using various shapes that represent the functional class of the gene product, while edges are displayed with various labels that describe the nature of the biological relationship between the nodes (see legend in the figure).

Figure 3

cDIA1R expression in the chick embryo. cDIA1R in situ hybridization was performed on whole-mount embryos at HH5 (A), 8 (B), 11 (C), 13 (D), 17 (E), 18 (F,G) and on a E10 brain cryosection (H). (B’,D’,E’,F’,F”) Sections of correspondent whole-mount embryos. (C1–C4) Regions of embryo in (C) at high magnification. cDIA1R expression starts to be detected at HH5 (A) in the extraembryonic mesoderm that will form the yolk sac blood islands at later stages (B,B’,C4). In HH11 embryos (C), cDIA1R is also expressed in the endocardium (arrow in C1), in the developing head vasculature (arrowheads in C1 and C2), in cells associated with the dorsal mid- and hindbrain (asterisks in C2) and in the dorsal aorta region (DA; C3). At later stages (D–H), cDIA1R expression is detected in most blood vessels of the embryo, such as the dorsal aorta (DA in D,D’), intersomitic vessels (E), head vasculature (E’,F,F’,F”) and allantois (G). Higher intensity is found in particular blood vessel cells that resemble hemogenic endothelial cells (D’,E’,F”; arrowheads). In the brain neuroepithelium (H), cDIA1R is detected both in the neurovasculature (arrows) and in isolated cells that may be microglial cells (arrowheads). Scale bars: 50 μm in B’,E’,F”; 100 μm in D’,F’.