Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development

Abstract

Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors.

Fig 1. AC3 expression in adult mouse cilia.

(A) Brain: note the long AC3 positive cilia in grey matter and the absence of AC3 positive cilia in white matter (corpus callosum). (B) Peripheral layers of the olfactory bulb: note the short AC3-positive cilia in the olfactory nerve layer (arrow) as compared to the long cilia of cells around the olfactory glomeruli (arrow head). (C) Seminiferous tubule: AC3 is present in the developing acrosome of round spermatids. Abbreviations: GM, grey matter; WM, white matter; ONL, olfactory nerve layer; G, glomerulus; GL, glomerular layer; PL, plexiform layer.

Fig 2. AC3-positive cilia in human and mouse tissues of mesenchymal origin during development.

(A-D) Tissue sections from a GW12 human fetus. (A) Meckel’s cartilage: short AC3-positive cilia in perichondral cells and chondroblasts. (B) Bone trabecula: AC3-positive cilia are present in osteoblasts (arrow) and osteocytes (arrow head). (C) Myotubes. (D) Mesenchymal cells. (E, F) Adjacent sections of a mouse tooth at the bell stage. (E) Hematoxylin-eosin staining. (F) AC3 immunohistochemistry shows positive cilia on odontoblasts (o) and peripheral cells of the enamel organ (eo) but not in ameloblasts (a). Abbreviations: AC3: adenylate cyclase type III; HE: Hematoxylin-eosin.

Fig 3. AC3-positive cilia in human epithelia.

(A) Olfactory epithelium in GW8 embryo; AC3-positive cilia on olfactory neurons (arrows). (B-C) Inner ear of a GW8 embryo. (B) Section through the endolymphatic duct (arrow) and semicircular ducts (arrow head). (C) Adjacent section showing AC3-positive primary cilia of apical epithelial cells lining the endolymphatic duct. (D-F) Kidney of a GW12 fetus. (D) Primary cilia are present on epithelial cells of the glomerulus and the distal tubule as well as on mesenchymal cells of the floculus and in interstitial cells. (E-F) AC3 is absent from apical epithelial cilia but present in mesenchymal cells cilia of the glomerular floculus and in the interstitium (F is a high magnification of E). (G-I) Pancreas of a GW12 fetus. (G) Hematoxylin-eosin stained section; only ducts but not secretory acini are present at this developmental stage. (H) Apical cilia of pancreatic duct are very long as compared to interstitial cells cilia. (I) Only cilia of cells around the ducts are AC3-positive (arrows). (J-L) liver of a GW12 fetus. (J) Portal space surrounded by the ductal plate (arrow) and small biliary ducts (arrow head). (K) Ductal plate cells (arrow) and the small biliary ducts cells (arrow head) have long primary cilia. (L) Absence of AC3 in epithelial cells cilium. (M-O) Stratified epithelia (M) Respiratory epithelium AC3 positive cilia of basal cells (arrows). (N-O) Oral epithelium. (N) Cilia in basal cells (arrow); presence of small nerve fibers in the lamina propria (arrow heads). (O) The cilia of basal cells are AC3 positive. Abbreviations: AC3: adenylate cyclase type III; Ac Tub: acetylated-tubulin; HE: Hematoxylin-eosin.

Fig 4. AC3-positive cilia in fibroblast culture.

There is an increasing number of fibroblasts extending an AC3 positive cilium with increasing cell confluence. Values represent means ± SEM (*p<0.05; **p<0.01). Solid lines concern AC3 and dotted lines concern acetylated tubulin. Abbreviations: AC3: adenylate cyclase type III; Ac Tub: acetylated-tubulin.

Fig 5. Endothelial cells cilia.

(A) Short AC3-positive cilia (arrows) on some endothelial cells in a GW8 human embryo. (B-E) Endothelial cells culture. (B) One cell displays an acetylated tubulin-positive primary cilium. (C) Three adjacent cells display an AC3-positive cilium. (D) One cell extends a primary cilium (arrow) from the gamma tubulin-labelled centrosome. (E) Only one of the three cells displays a primary cilium which is AC3 positive (arrow). Abbreviations: AC3: adenylate cyclase type III; Ac Tub: acetylated-tubulin; γTub: gamma tubulin.