Development of the human choriocapillaris

Abstract

Vasculogenesis and/or angiogenesis are thought to be the major mechanisms for new vessel formation during development. A third mechanism, haemo-vasculogenesis, has been described in which blood vessel and blood cells (haematopoiesis (expression of CD34(+)) and erythropoiesis (presence of epsilon chain of haemoglobin or Hb-epsilon(+))) differentiate from a common precursor, the haemangioblast. This review describes the mechanism(s) for development of human choroidal vascular from 6 until 22 weeks gestation (WG). Endothelial cell or EC (CD31, CD34, CD39, VEGFR-2) and angioblast (CD39, VEGFR-2) markers were present in choriocapillaris (CC) by 7 WG through 22 WG. From 6 to 8 WG, many erythroblasts (nucleated Hb-epsilon(+) RBCs) were observed in the CC layer. Erythroblasts (Hb-epsilon(+)) were also positive for CD34, CD31, and/or VEGFR-2. Proliferation of vascular cells (Ki67+), suggesting angiogenesis, was not observed until 12 WG. TEM analysis demonstrated that CC was structurally immature even at 11 WG: no basement membrane, absence of pericytes, and poorly formed lumens that were filled with filopodia. Contiguous fenestrations and significant PV-1 (protein in diaphragms of fenestrations) were not observed until 21-22 WG. Smooth muscle actin was prominent at 20 WG and the maturation of pericytes was confirmed by TEM. Therefore, the embryonic CC appears to form initially by haemo-vasculogenesis (Hb-epsilon(+)/CD31(+) cells), whereas angiogenesis (CD34(+)/Ki67(+)) appears to be the mode of intermediate and large choroidal vessel development later in the foetus. Contiguous fenestrations, mature pericytes, and EC basal lamina occur late in development, around 22 WG, which coincides with photoreceptors developing inner segments.

Figure 1

Haemo-vasculogenesis in cross-sections of 6.5 WG foetal choroid (a–f) and a schematic representation of this process (g). In the choriocapillaris (CC) layer, erythroblasts (bright pink cytoplasm) can form a solid cord-like structures (double arrow) without a lumen (a). Erythroblasts, haematopoetic and vascular cells develop in situ with erythroblasts sometimes forming lumen (arrow) (d). Eventually, the outer cells become primarily ECs and the inner cells become primarily erythroblasts (f). Free erythroblasts (arrowheads in d–f) are present in stroma of choroid. (g) A schematic of haemo-vasculogenesis in CC where islands of progenitors are then united by other progenitors eventually yielding a chicken-wire pattern that will become lobular in the adult CC. (Scale bar = 10 μm; (a–f) Giemsa stained JB4 sections).

Figure 2

Co-localization of erythroid (ε haemoglobin, Hb-ε) and endothelial markers (CD31 and VEGFR-2 or FLK-1) in developing choriocapillaris (CC). (a–d) CD31 (red) and Hb-ε (green) are co-localized in cells of the developing CC (arrows) and single cells within the choroidal stroma (arrowhead). (e–h) FLK-1 and Hb-ε co-expression in cells lining a developing lumen (arrows) and in cells located outside of the structure (arrowhead). (Scale bars = 10 μm; counterstained with DAPI, blue).

Figure 3

CD31 immunolabelling of choroidal sections from foetal eyes at 6 WG (a), 12 WG (b), 16 WG (c), and 20 WG (d). At 6 WG (a), only a highly cellular rudimentary choriocapillaris (CC) with poorly defined lumen (arrow) present. At 12 WG (b), vessels are diving from the CC into the deeper choroid (arrowhead). By 16 WG (c), well-defined CC lumens are present (arrow) and a network of medium-size deeper blood vessels has formed (arrowhead). At 22 WG (d), the CC (arrow), medium-size vessels of the Sattler’s layer (arrowhead), and the larger outer blood vessels (open arrow) are all present. (Scale bar = 30 μm).

Figure 4

CD39 immunolabelled flat choroids showing the CC pattern (a, b) and TEM images showing the structure of developing CC (c–f). At 9 WG (a), CD39-positive cells are organized into highly cellular solid cord-like structures without apparent lumen. By 12 WG (b), the capillaries have thinned, lumen have formed, and cellularity has decreased markedly. (c) In ultrathin sections from 11 WG choroid, plump EC nuclei (e) with condensed chromatin line a slit-like lumen (asterisk). Cytoplasmic extensions (arrow) projected into the lumenal space and in some cases made intimate contact with erythroblasts. Perivascular cells, putative pericytes (p), had ultrastructural features identical to the immature ECs. (d) In sections from a 16 WG choroid, lumenal spaces were more apparent (asterisk), the EC nuclei had finer chromatin, were reduced in volume, and had decreased cytoplasmic projections. (e) By 22 WG, lumens were broad and flat, ECs had thinned and definitive pericytes (p) were present on the outer surface of the capillaries. At this stage of development, fenestrations were present along inner aspect of the CC. (f) Fenestrations on the retinal side of a CC lumen at 22 WG. (Scale bars = 20 μm (a, b); 2 μm (c, d); 4 μm (e); and 50 nm (f)).