A model to depict the role of Eng during early hematopoiesis. (A) E7.5 Eng⁺-sorted cells were cultured in serum-free hematopoietic medium containing SCF, IL-3, and Flt3L (HE) in the presence or absence of BMP2 (10 ng/mL), BMP4 (25 ng/mL), or TGFβ1 (25 ng/mL). Representative images of hematopoietic outgrowths obtained from each culture condition (10-day cultures) are showed at similar magnification (10× objective). Expansion of hematopoietic cells occurred only when members of TGFβ superfamily were added to the basic medium containing HE cytokines, particularly BMP4. Eng⁺ cells survived but did not proliferate in the absence of BMP2/4 or TGFβ1. Representative data from at least 2 independent experiments are shown. (B) Cytologic examination of outgrowths obtained from Eng⁺ sorted cells, cultured for 5 days in HE medium in the presence or absence of BMP2, BMP4, or TGFβ1 demonstrates the hematopoietic nature of these cells. Representative cytospins of 2 independent experiments. (C) Number of hematopoietic progenitors in Eng⁺ cell cultures after 4 days of expansion in HE medium with or without BMP2, BMP4, and TGFβ1. Bars represent the average number of colonies from 4 independent experiments. Error bars represent the SEM for each type of colony. (D) Schematic representation of an E7.5 embryo (left panel) shows the localization of Eng⁺ cells during early development. E⁺F⁺ and E⁺F⁻ cells are represented in yellow and orange, respectively. Red round cells represent primitive erythrocytes. During the formation of BIs at E7.5, E⁺F⁻ cells (right panel, orange), possibly from the chorion, secrete BMP4, which through autocrine signaling may lead to activation of SMAD1 after binding to the Eng/ALK3/BMPRII receptor complex, with subsequent activation of the downstream genes Msx1 and Id2. BMP4 may also have a paracrine effect in E⁺F⁺ cells (right panel, yellow), early mesodermal progenitors present in the extraembryonic region that are endowed with hematopoietic and endothelial potential, because these cells express Eng/ALK3/BMPRII receptors and high levels of Gata2, Scl, and Lmo2, also target genes of BMP4. The finding that E⁺F⁺ cells display high levels of TGFβ1 and BMP2, as well as their respective receptor complexes Eng/ALK1/TGFBRII and Eng/ALK3/BMPRII, suggest a potential autocrine loop, because high levels of pSMAD1/5 (blue circles) and c-Myc and Cyclin D1 are detected in these cells. The important role played by Eng in early hematopoiesis may be the modulation of the activation of TGFβ1 and BMP2/4 signaling pathways. The gray dashed arrow indicates a possible secreted molecule and the gray solid arrow represents a potential signaling pathway.