CD34 Expression Patterns During Early Mouse Development Are Related to Modes of Blood Vessel Formation and Reveal Additional Sites of Hematopoiesis by Heather B. Wood, Gillian May, Lyn Healy, Tariq Enver, and Gillian M. Morriss-Kay Blood Volume 90(6):2300-2311 September 15, 1997 ©1997 by American Society of Hematology

Expression of CD34 mRNA during early cardiovascular development in the mouse embryo. Expression of CD34 mRNA during early cardiovascular development in the mouse embryo. Cranial/anterior is to the right in (A) and (B) (n, cranial neural fold). (A) 1-2–somite headfold stage: transcripts are detectable in two diffuse lines (arrowed), corresponding to the two presumptive dorsal aortae, continuous cranially with the presumptive paired endocardial heart tubes (arrowhead). (B) 3-somite stage: the paired dorsal aortae (da) are now clearly defined in the anterior half of the embryo, and are continuous with diffuse lines of stained cells, presumably pre-endothelial cells (arrowed), which join at the junction of the primitive streak (ps) and allantois; there is diffuse staining in the allantois and yolk sac (y). (C) 7-somite stage: caudally, the two dorsal aortae (da) are continuous with the omphalomesenteric artery (om) which is also stained; intersomitic arteries (is) have begun to branch from each dorsal aorta; endocardial (en) staining is clear in the ventricle and truncus arteriosus, which is continuous with the first aortic arch (arrow); there are also scattered stained cells within the cranial mesenchyme (c). (D) 9-somite stage: CD34 transcripts are now more abundant in the cranial mesenchyme (c) and in the endothelial lining (en) of the ventricle and truncus arteriosus of the heart; there are clear patches of stained cells in the yolk sac (y). (E) 14-somite stage: CD34 transcripts are abundant in the umbilical artery (ua), allantoic mesenchyme (al), umbilical vein (arrows) and proximal segment of the vitelline arteries (va); in the head, cranial mesenchymal cells expressing CD34 are beginning to condense around the developing eye close to the internal carotid artery (arrow); the endothelium of the first aortic arch artery (1) is stained, but transcript levels are low in the second aortic arch (2) and undetectable in the third aortic arch (3) or in the aortic sac that joins the second and third aortic arches; transcript levels are lower in the anterior cardinal vein (ac) and common cardinal vein (cc) than in the dorsal aorta (da) and umbilical vein (uv), and undetectable in the vitelline vein (vv); they are low in the ventricle (v), atrium (not visible) and sinus venosus (s), but high in the truncus arteriosus (ta). (F ) Day 9, 18-somite stage: transcripts are now present in the aortic sac (a) and all aortic arches, but in general are more abundant in capillary networks around the developing sense organs than in the larger vessels. (G) Day 10 embryo head: the hyaloid artery (arrow) is stained, but in general transcript levels are decreased in the head, being confined to capillary networks around the eye and brain and within the facial mesenchyme. e, eye; n, nasal pit; o, otic pit. Scale bars = 100 μm Heather B. Wood et al. Blood 1997;90:2300-2311 ©1997 by American Society of Hematology

Immunohistochemical detection of CD34 glycoprotein in the developing embryo. Immunohistochemical detection of CD34 glycoprotein in the developing embryo. (A) Transverse section of the cranial region of an 8-somite stage embryo, showing staining in the two dorsal aortae (da); staining is barely detectable in the primary head vein (v). (B) Dorsal aorta in the trunk region of an 8-somite embryo in longitudinal section: the endothelium expresses CD34. (C) Transverse section of a 10-somite stage embryo, showing CD34 expression in the endothelium of the dorsal aorta and in blood cells in the lumen; staining is not present in the posterior cardinal vein (v); slight staining of dorsal gut (g) epithelium. (D) Longitudinal section through the trunk of a 12-somite stage embryo: staining of the endothelium and blood cells of the dorsal aorta (da), in adjacent mesenchymal cells (open arrow), and in the neuroepithelium (n). (E) Transverse section (dorsal/ventral is left/right, medial/lateral is top/bottom) of one of the dorsal aortae (da) of a day 10 embryo, showing CD34 expression on the endothelial surface and in discrete rounded cells in the adjacent mesenchyme (arrows); these are probably hematopoietic cells. The posterior cardinal vein (v) is unstained. (F ) Fused dorsal aorta (da) of a day 10 embryo, showing staining on the endothelial surface and also on large (probably hematopoietic) cells attached to the vessel walls. There is also staining in many small vessels within the nephrogenic mesenchyme, and of the posterior cardinal veins (v), which appear to be generating the capillary sprouts. (G) Day 10 embryo, showing CD34+ precapillary sprouts (arrow) surrounding the tracheal diverticulum (t) and, to a lesser extent, the oesophagus (g). (H) Allantois of a 10-somite embryo, showing CD34 expression in the walls and blood cells of a formed vessel (large arrow) and in prevascular mesenchyme (small arrows). (I) Day 10 liver, showing staining in hematopoietic cells but not on the endothelium of the sinusoids (s). c, coelom; g, gut; ge, gonadal epithelium; m, mesonephric duct; n, neural epithelium. Scale bars = 50 μm Heather B. Wood et al. Blood 1997;90:2300-2311 ©1997 by American Society of Hematology

Expression of CD34 mRNA in the developing mouse yolk sac and allantois. Expression of CD34 mRNA in the developing mouse yolk sac and allantois. (A) 4-5–somite stage: many of the stained cells are elongated and surround unstained tissue, suggesting that they are endothelial cells lying around the periphery of the blood islands. (B) 12-somite stage: endothelial staining is weaker but the rounded haematopoietic cells retain CD34 expression. (C) Day 9, 20-somite stage: the only CD34 expression in the yolk sac is in the vitelline artery at the point that it emerges from the embryo. (D) Vitelline artery at the 20-somite stage at a higher magnification: the stained endothelial cells are discontinuous. (E) Day 10 embryo (around 30 somites), showing the points of emergence of the umbilical (u) and vitelline (v) vessels from the embryo. (F ) Day 10 umbilical vessels dissected away from the embryo, showing foci of CD34+ staining; the allantoic capillaries within the chorioallantoic placenta are strongly stained. (G and H) Day 10 vitelline artery dissected away from the embryo, showing foci of CD34+ cells at the proximal end (left) only. Scale bars = 100 μm Heather B. Wood et al. Blood 1997;90:2300-2311 ©1997 by American Society of Hematology

Expression of CD34 glycoprotein in the developing yolk sac and extraembryonic blood vessels. Expression of CD34 glycoprotein in the developing yolk sac and extraembryonic blood vessels. (A) Yolk sac from a 4-somite stage embryo: staining appears to be confined mainly to the endodermal layer (e); light staining (arrow) is present between two blood islands in the mesodermal layer. (B) Yolk sac from a 6-somite stage embryo: there is staining in the endodermal layer and in scattered cells around the periphery of the blood islands (arrows). (C) Yolk sac and allantois of an 8-somite embryo: CD34 is expressed in the endoderm (e) and around the periphery of the blood islands (arrows), but not in the hematopoietic cells nor in the allantoic mesenchyme (al) except for small blood vessels (open arrow). (D) Yolk sac from a 10-somite embryo: staining of the endothelium of the vitelline blood vessels (v) is comparable with that of the dorsal aorta (da); large cells (arrowed) within the vessels are also stained. (E) Yolk sac of an 18-somite stage day 9 embryo, showing less prominent staining in the vascular endothelium and a subset of strongly stained cells (arrows) within the lumina of the vessels. (F ) Day 10 yolk sac, showing CD34 expression on the endothelium of flattened vessels; large stained cells are absent. (G) Proximal portion of the vitelline artery, showing CD34+ sites of possible haematopoietic activity. (H) Umbilical artery, showing CD34+ hematopoietic cells (arrow) and a thickened, possibly haematopoietic region. Scale bars (A through F on F ) = 50 μm. Heather B. Wood et al. Blood 1997;90:2300-2311 ©1997 by American Society of Hematology

Diagrammatic summary indicating the intensity of staining for CD34 RNA transcripts and protein in developing blood vessels, indicating the relationship between staining intensity (as indicated by depth of shading) and the mode of blood vessel formation. Diagrammatic summary indicating the intensity of staining for CD34 RNA transcripts and protein in developing blood vessels, indicating the relationship between staining intensity (as indicated by depth of shading) and the mode of blood vessel formation. In general, CD34 expression is strong in pre-endothelial cells, in vessels formed by condensation of pre-endothelial cells (ie, by vasculogenesis), particularly the dorsal aortae. Equally strong staining is observed in vessels forming by capillary sprouting, but the larger vessels of the aortic arch region are not strongly stained during their formation even though their origin can be described as angiogenetic. Vessels formed by the coalescence of networks of smaller vessels showed low or undetectable levels of CD34 staining. Heather B. Wood et al. Blood 1997;90:2300-2311 ©1997 by American Society of Hematology