Neural Crest Cells Contribute an Astrocyte-like Glial Population to the Spleen

Abstract

Neural crest cells (NCC) are multi-potent cells of ectodermal origin that colonize diverse organs, including the gastrointestinal tract to form the enteric nervous system (ENS) and hematopoietic organs (bone marrow, thymus) where they participate in lymphocyte trafficking. Recent studies have implicated the spleen as an anatomic site for integration of inflammatory signals from the intestine with efferent neural inputs. We have previously observed alterations in splenic lymphocyte subsets in animals with defective migration of NCC that model Hirschsprung's disease, leading us to hypothesize that there may be a direct cellular contribution of NCC to the spleen. Here, we demonstrate that NCC colonize the spleen during embryogenesis and persist into adulthood. Splenic NCC display markers indicating a glial lineage and are arranged anatomically adjacent to blood vessels, pericytes and nerves, suggesting an astrocyte-like phenotype. Finally, we identify similar neural-crest derived cells in both the avian and non-human primate spleen, showing evolutionary conservation of these cells.

Figure 1. Embryonic and postnatal hindgut and spleens of EdnrB^NCC+/− and EdnrB^NCC−/− animals.

(A) tdTomato visualization of NCC in the small intestine and colon shows delayed colonization of the colon in EdnrB^NCC−/− animals compared to EdnrB^NCC+/−, with the migratory wavefront of NCC marked by white arrows at E14.5. (B) Aganglionosis in the distal colon of EdnrB^NCC−/− animals at P21 causes functional obstruction (marked by white arrowhead). Normal, pelleted stool is seen in the distal EdnrB^NCC+/− colon. (C) Reduced splenic size of EdnrB^NCC−/− compared to EdnrB^NCC+/− animals at P21. (D) tdTomato expressing NCC in EdnrB^NCC+/− and EdnrB^NCC−/− spleens at P21. (E) Spleens were harvested from EdnrB^NCC+/− and EdnrB^NCC−/− animals at P0, P9 and P18 and weighed. (*p < 0.05). Scale bars: A 400 μm, B 1 cm, C 4 mm and D 100 μm.

Figure 2. Identification of entry of tdTomato expressing NCC into the spleen.

(A) Whole mount preparation of the spleen and hilar structures at E16.5 shows numerous NCC on the hilum and a small number within the spleen. (B) Upper panel: 16 μm section showing tdTomato NCC entering the spleen (Sp) along the hilum (splenic artery SA, stomach St) express EdnrB (yellow in merged panels). Lower Panel: High magnification of boxed region in upper panel. (C) At E17.5, tdTomato NCC enter the spleen from the hilum and colonize the organ. (C’) High magnification view of a single splenic NCC at E17.5. (D) By P0, large numbers of NCC have colonized the spleen in an arborized pattern. Arrowheads (white) highlight individual splenic NCC. Scale bars: A 200 μm, B 50 μm, C 200 μm, C’ 20 μm, D 200 μm.

Figure 3. NCC along the splenic hilum at E16.5 express glial markers.

(A) tdTomato NCC are located near, but are distinct from CD31 (endothelial cells) and (B) PDGFRβ (pericytes) which surround blood vessels. (C) tdTomato NCC are associated with TuJ1 (nerve fibers). (D) At E16.5, most tdTomato NCC express BLBP (early glial lineage) and (E) S100-β (glial lineage). Arrowheads (white) highlight double-labeled cells. Scale bar: 50 μm.

Figure 4. Splenic NCC within the spleen at P21 express glial markers.

(A) Within the spleen, tdTomato NCC are present along blood vessels (CD31). (B) tdTomato NCC are associated with pericytes (PDGFRβ) and (C) nerve fibers (TuJ1). (D) At P21 all tdTomato NCC express S100-β (glial lineage). Scale bar: 50 μm.

Figure 5. NCC are found in the spleens of avians and non-human primates.

(A) NCC (HNK1 red) are located around blood vessels (endogenous GFP, green) and co-express GFAP (glial lineage, white) in the Day 19 embryonic chick spleen. (B) Top: NCC (p75, red) surround blood vessels (CD31, green) in the juvenile non-human primate spleen. Middle: Glial cells (S100-β, red) are located in a similar position to NCC. Bottom: Glial cells (S100-β, red) are found in proximity to TuJ1+ nerve fibers (green). See also Supplementary movie. Scale bar: 50 μm.

Figure 6. Expression of S1P1 is reduced in EdnrB^NCC−/− spleen.

Relative expression of S1P1 is reduced in EdnrB^NCC−/− versus EdnrB^NCC+/− spleens at P21.