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Gen Comp Endocrinol
2014 Sep 01;205:242-50. doi: 10.1016/j.ygcen.2014.05.014.
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Early expression of aromatase and the membrane estrogen receptor GPER in neuromasts reveals a role for estrogens in the development of the frog lateral line system.
Hamilton CK
,
Navarro-Martin L
,
Neufeld M
,
Basak A
,
Trudeau VL
.
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Estrogens and their receptors are present at very early stages of vertebrate embryogenesis before gonadal tissues are formed. However, the cellular source and the function of estrogens in embryogenesis remain major questions in developmental endocrinology. We demonstrate the presence of estrogen-synthesizing enzyme aromatase and G protein-coupled estrogen receptor (GPER) proteins throughout early embryogenesis in the model organism, Silurana tropicalis. We provide the first evidence of aromatase in the vertebrate lateral line. High levels of aromatase were detected in the mantle cells of neuromasts, the mechanosensory units of the lateral line, which persisted throughout the course of development (Nieuwkoop and Faber stages 34-47). We show that GPER is expressed in both the accessory and hair cells. Pharmacological activation of GPER with the agonist G-1 disrupted neuromast development and migration. Future study of this novel estrogen system in the amphibian lateral line may shed light on similar systems such as the mammalian inner ear.
Fig. 1.
Aromatase and GPER are expressed throughout early S. tropicalis embryogenesis. Western blots and corresponding densitometry analysis demonstrate an overall increase in the amount of (A) aromatase and (B) GPER around NF34 (significant only in the case of aromatase). The densitometric analysis is reported as an arbitrary value relative to a positive control sample (ovarian tissue) ± SEM. For each stage, animals were pooled in groups of 20 (r = 6) prior to protein isolation.
Fig. 2.
Neuromast localization using the hair cell specific dye, FM1–43. Examples of FM1–43 stained neuromasts (green) are depicted with the thick white arrows. Only two neuromasts have developed at NF34 (A) and are present at the dorsal margin of the eye (e). By NF47 (B) the neuromasts have developed and migrated along the head (B). (C) Following exposure to 200 μM neomycin, the hair cells are obliterated and are no longer stained with FM1–43. cg = cement gland, scale bar = 200 μm.
Fig. 3.
GPER (top row, red), aromatase (middle row, red) and the glial cell marker GFAP (bottom row, red) expression in developing neuromasts of NF34 (first column) and NF47 (second and third columns) larvae. All larvae were counter-stained with the nuclear dye Hoechst 33342 (blue). Also shown in NF34 larvae is the result of the co-staining of FM1–43 (green) dye which specifically labels hair cells. In NF34 larvae (A, D, G), GPER, aromatase and GFAP are present in developing neuromasts including those which are FM1–43 negative (arrowheads). In NF47 tadpoles, the expression of GPER (B, C) and aromatase (E, F) is primarily in the overlaying mantle cells of the neuromasts, while the expression of GFAP (H, I) is primary detected in the underlying support cells. There is also some expression of GPER in the hair cells. Note the strong expression of the glial cell marker GFAP in the microvilli of the support cells which extend out of the apical regions of the support cells (asterisk). arom = aromatase, scale bar = 25 μM.
Fig. 4.
Double-staining of aromatase, GPER or GFAP with FM1–43 in NF47 tadpoles imaged with confocal microscopy (A–D) or light microscopy (E). (A) Superficial plane of neuromast shows that aromatase (red) is expressed primarily in the apical region of the mantle cells. (B) Mid-section plane of neuromast shows that aromatase (red) is not co-localized with FM1–43 (green) and therefore is not present in the hair cells. (C) Superficial plane of neuromast shows GPER (red) is expressed in the mantle cells. (D) Mid-section plane of neuromast shows that GPER (red) is co-localized (yellow) with FM1–43 (green), thus it is also present in the hair cells. (E) The glial-cell marker GFAP (red), is expressed primarily in the supporting cells, particularly in the microvilli and is not co-localized with FM1–43 (green). Blue = Hoechst 33342 nuclear stain. Scale bar = 25 μm.
Fig. 5.
Whole-mount immunofluorescence of neuromasts of NF47 larvae double-stained with GPER (red, first row) or aromatase (red, bottom row) and the afferent nerve specific antibody ZN-12 (green, first column) or the glial cell specific marker GFAP (green, second column). (A) GPER (red) is expressed primarily in the mantle cells of the neuromast and shows no co-localization with ZN-12 (green) indicating that it is not present in the nerve fibers that innervate the neuromast. (B) GPER (red) is expressed in the mantle cells and shows little co-localization with GFAP (green) which is expressed predominately in the supporting cells. (C) Aromatase (red) is expressed in the mantle cells and shows no co-localization in the afferent nerve fibers labeled with ZN-12 (green). (D) Aromatase (red) is expressed in the mantle cells and shows little co-localization in the supporting cells which are labeled with GFAP (green). Scale bars = 25 μm.
Fig. 6.
Exposure of embryos to varying doses of G-1 causes (A, B) severe developmental abnormalities (shortened total length, axial curvature, pericardial/yolk sac edema, craniofacial abnormalities) and (B) a decrease in the total number of neuromasts present in the lateral line, in a dose-dependent manner and (C) a decrease in the total number of neuromasts present in the lateral line, in a dose-dependent manner.
Fig. 7.
Diagrammatic representation of the expression of aromatase, GPER and GFAP in the neuromast and the possible autocrine and paracrine actions of estradiol-17β (E2) in this system; E2 is synthesized by aromatase in the mantle cells and acts on GPER in both the mantle and the hair cells to regulate cell proliferation (Image adapted from Ghysen and Dambly-Chaudiere (2007)).
