Endothelial cell lineages of the heart

Abstract

During early gastrulation, vertebrate embryos begin to produce endothelial cells (ECs) from the mesoderm. ECs first form primitive vascular plexus de novo and later differentiate into arterial, venous, capillary, and lymphatic ECs. In the heart, the five distinct EC types (endocardial, coronary arterial, venous, capillary, and lymphatic) have distinct phenotypes. For example, coronary ECs establish a typical vessel network throughout the myocardium, whereas endocardial ECs form a large epithelial sheet with no angiogenic sprouting into the myocardium. Neither coronary arteries, veins, and capillaries, nor lymphatic vessels fuse with the endocardium or open to the heart chamber. The developmental stage during which the specific phenotype of each cardiac EC type is determined remains unclear. The mechanisms involved in EC commitment and diversity can however be more precisely defined by tracking the migratory patterns and lineage decisions of the precursors of cardiac ECs.

Fig. 1

Schematic illustration of the heart field mesoderm (cm, black), paraxial mesoderm (pam, light gray) and lateral plate mesoderm (lpm, dark gray) in zebrafish (left panel) and chick (middle panel) gastrula stage embryos. an-ve: the animal-vegital axis, v-d: the ventral-dorsal axis, and a-p: the anterior-posterior axis, ps: the primitive streak. Right panel: Lineage relationships between multiple endothelial cell types generated from mesoderm. As three germ layers form via gastrulation, endocardial ECs differentiate from the heart field mesoderm, while coronary vasculature, including ECs, differentiate from a distinct mesodermal tissue, the proepicardium. Non-cardiac arterial, venous and capillary ECs are generated from the lateral plate and somatic mesoderm. Lymphatic ECs appear to differentiate from venous ECs during budding off.

Fig 2

Expression pattern of PE and liver-bud markers in chick embryos. (A) Single whole-mount in situ hybridization for a PE marker Wt1 (purple). (B) Double in situ hybridization for Wt1 (purple) and a liver-bud marker Hex (green). (C) Section from an embryo double hybridized for a PE marker Cfc (purple) and a liver bud marker Hex (green). Experimental procedures as described elsewhere (Ishii et al., 2007).

Fig. 3. Detection of EC differentiation in the developing PE (A–C) and in clone density culture of PE cells (D–G)

(A) A transverse section through the PE, immunostained for an EC marker QH1 (red) and a mesothelial PE cell marker Wt1 (green). Dorsal is up. DAPI was used to visualize nuclei (blue). (B,C) High magnification of a boxed region in A. While many ECs are found in the mesenchymal core (asterisk) of the PE, some ECs are also detected in the Wt1-positive mesothelial component of the PE (arrowheads). (D) Single PE cell cultured for 6hr at the clonal density. The quail PE cells were dissociated by pipetting after treatment with collagenase and with trypsin/EDTA. The cells were cultured in a growth medium (M199 with 0.1 mg/ml heparin, 0.05 mg/ml endothelial cell growth supplement and 10% fetal bovine serum) on a plastic dish. (E) Daughter cells of (D) after 36 hr of plating. (F,G) Cluster of PE cells triple stained for EC marker (QH1; red), smooth muscle marker (smooth muscle alpha-actin; green) and DAPI for nuclei (blue).