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Nicotinic receptor alpha7 expression during mouse adrenal gland development

PLoS ONE (2014) 9(8)
DOI: 10.1371/journal.pone.0103861
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Abstract

The nicotinic acetylcholine receptor alpha 7 (α7) is a ligand-activated ion channel that contributes to a diversity of cellular processes involved in development, neurotransmission and inflammation. In this report the expression of α7 was examined in the mouse developing and adult adrenal gland that expresses a green fluorescent protein (GFP) reporter as a bi-cistronic extension of the endogenous α7 transcript (α7 G). At embryonic day 12.5 (E12.5) α7G expression was associated with the suprarenal ganglion and precursor cells of the adrenal gland. The α7G cells are catecholaminergic chromaffin cells as reflected by their progressive increase in the co-expression of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) that is complete by E18.5. In the adult, α7G expression is limited to a subset of chromaffin cells in the adrenal medulla that cluster near the border with the adrenal cortex. These chromaffin cells co-express α7G, TH and DBH, but they lack phenylethanolamine N-methyltransferase (PNMT) consistent with only norepinephrine (NE) synthesis. These cell groups appear to be preferentially innervated by pre-ganglionic afferents identified by the neurotrophin receptor p75. No afferents identified by beta-III tubulin, neurofilament proteins or p75 co-expressed α7G. Occasional α7G cells in the pre-E14.5 embryos express neuronal markers consistent with intrinsic ganglion cells and in the adult some α7 G cells co-express glutamic acid decarboxylase. The transient expression of α7 during adrenal gland development and its prominent co-expression by a subset of NE chromaffin cells in the adult suggests that the α7 receptor contributes to multiple aspects of adrenal gland development and function that persist into adulthood.

Figures

  • Figure 1. Specificity of a7G detection in the adrenal gland. A. Transverse sections from a post-natal day 7 (P7) a7G mouse adrenal gland. AntiGFP (Primary) labeling using the immuno-peroxide method reveals cells localized throughout the adrenal medulla (med) that exhibit expression. Cells in the adrenal cortex (ctx) were not labeled. On the right is a similar section that was processed but the antibody to GFP was omitted (No Primary). B. Transverse sections from an adult a7G adrenal gland processed for GFP immunoreactivity as in A. On the right an adjacent section was processed without addition of anti-GFP primary antibody. Arrow heads identify cells immunopositive for GFP signal which tend to be in clusters near the boundary of the medulla and cortex (darkly colored in this preparation). C. Increased magnification of the adult adrenal gland medullary region showing immune-reactivity towards GFP. The arrows point to cell clusters expressing GFP indicative of a7G expression. The section to the right was again processed the same way but with omission of primary antibody. D. Transverse sections from an adult a7G mouse adrenal gland were processed using immunohistochemistry to reveal anti-GFP immunofluorescence. On the right is a section processed without addition of anti-GFP primary antibody. Arrows identify a7G cells immunopositive for GFP signal. The white oval outline approximates the boundary between the med and ctx. Note the similarity between the immunofluorescence pattern of GFP expression in cell clusters near the medulla-cortex boundary that closely resemble the pattern of immune-staining seen in B and C using the immuno-peroxide method. E–H. Additional tests confirm the specificity for measurement of the a7G expression pattern in the adrenal gland. E. Sections were taken from E14.5 embryos of various genotypes related to a7 expression. First, immunofluorescence to a7G (a7GFP) is revealed using and anti-GFP primary antibody. The anti-GFP signal is prominently expressed in chromaffin cells of the developing adrenal medulla (med), but it is essentially absent from the adrenal cortex (ctx). The kidney is identified (ki). F. A similar section was prepared from an a7 knock-out (a7KO) adult mouse and processed for anti-GFP immunofluorescence. No detectable GFP signal was observed. G. Another independent method to measure a7 expression was tested in an E14.5 embryo from the a7Cre to a Rosa26-loxP(LacZ) reporter mouse cross (a7Cre:LacZ; for details see [28]). This embryo exhibits beta-galactosidase staining consistent with chromaffin cell expression. H. Sections prepared from E14.5 embryo of the a7Cre x Rosa26-LoxP(diphtheria toxin (DTA) cross to conditionally ablate (a7Cre:DTA; see [28]). Anti-GFP immunofluorescence detects no a7G and there is no defined adrenal medulla (compare with E). I. In an E18.5 a7G embryo the co-labeling for GFP (a7GFP; green) and the a7-associated hemagglutinin epitope tag (HA; see [28]) reveals GFP (green) that diffuses throughout the cytoplasm but does not enter the nucleus. The HA immunostaining (red) is located on the cell surface of the GFP expressing cells usually in a punctate pattern (identified by arrows). For A–H the Bar = 100 mm and for I the Bar = 30 mm. doi:10.1371/journal.pone.0103861.g001
  • Figure 2. Expression of a7G in the developing mouse adrenal gland. Sagittal sections of embryos at different stages of development were prepared and immunostained for a7G (anti-GFP, Methods) to reveal current a7 expression. A,B. E12.5 embryos show the developing adrenal gland (AG) adjacent to the kidney (ki). Immunostaining for GFP is seen in the AG, present in the developing adrenal gland (AG, arrow heads), the suprarenal ganglion (SRG), enteric neurons (arrows) and the ureteric buds (utb) of the kidney. At increased magnification (B), the GFP signal in the AG and SRG reveals a7G positive cells (arrow heads). C,D. The E14.5 AG begins to show consolidation of the cells expressing a7G (arrow head) and ongoing expression in the SRG. At greater magnification (D), these cells tend to be in aggregates and often appear to be of increased density in the more ventral aspect of the AG (arrow heads). E,F. An E16.5 AG and associated SRG stained for GFP. At this stage a7G expressing cells are in the medulla and few (if any) remain in the now recognizable capsule (cpsl) or ctx. Increased magnification (F) shows the majority of a7G stained cells are in welldefined aggregates within the medulla (arrow heads). G,H. At E18.5 a7G remains localized to well-defined cell groups in the med (arrow heads). The image on the right shows that at this stage there are also cells of reduced a7G intensity (asterisk) relative to those identified by the arrow heads. I,J. The post-natal day 7 (P7) adrenal gland. Cells expressing a7G tend to be aggregated into clusters that are localized towards the med-ctx border (arrow heads). This is particularly evident in (J) where the cell aggregates are visible while more centrally localized cells exhibit diminished or no a7G expression (asterisk). K,L. The adult adrenal gland a7G expression persisted in only a relatively few cells clustered into groups near the med-ctx boundary (arrow heads). The ‘spotted’ staining (asterisk) at this boundary interface and extending into the ctx is background fluorescence. Abbreviations: AG, adrenal gland; cpsl, adrenal capsule; ctx, adrenal cortex; ki, kidney; med, adrenal medulla; SRG, suprarenal ganglion; utb, ureteric bud. Scale bars = 100 mm (A,B,C,E,G,I,K) or 50 mm (D,F,H,J,L). doi:10.1371/journal.pone.0103861.g002
  • Figure 3. Coincident appearance of a7G and catecholamine markers during chromaffin cell development. The adrenal glands from a7G mice at the E12.5, E14.5, E16.5 and E18.5 developmental stages were co-stained for immunoreactivity to GFP (green) and in red either (A) tyrosine hydroxylase (TH) or (B) dopamine-beta-hydroxylase (DBH). C. Quantification of the a7G cells co-labeled with either TH or DBH for the developmental stages. For each point at least 3–5 sections from the adrenal glands were collected from 3–6 different animals and the results from each individual animal were then summarized. The error bars reflect +/2 standard error of the mean. Scale bar = 25 mm (E12.5, E14.5) or 50 mm (E16.5, E18.5). doi:10.1371/journal.pone.0103861.g003
  • Figure 4. Co-expression of a7G, catecholamine markers and GAD65/67 in the adult adrenal medulla. Adult adrenal medulla immunostained for a7G or DBH, PNMT or GAD 65/67 as identified. Cells expressing a7G as measured by GFP immunofluorescence (green) are present and they tend to be clustered towards the adrenal medulla-cortex boundary (arrow). Some a7G expression is localized to cells which are smaller and more solitary but exhibit a particularly strong GFP signal (arrow head). As shown most a7G cells co-express DBH (arrow), but the more solitary cells do not (arrow head). In contrast co-expression of a7G and PNMT has not been observed. The staining for GAD65/67 is largely discordant from that of a7G although rare cells expressing both markers are identified (arrow). The extensive staining by GAD65/67 can also be found in some afferent fibers that are often associated with cells in clusters that express a7G as is seen at greater magnification (cluster identified by the arrow). Scale bar = 50 mm. doi:10.1371/journal.pone.0103861.g004
  • Figure 5. Adrenal gland innervation and expression of a7G. Adrenal glands at various times of development as indicated were labeled for coexpression of a7G as measured by anti-GFP immunofluorescence (a7GFP, green) and various peripheral nerve markers as indicated (red). A) The same section from Figure 2a for the E12.5 adrenal gland is shown with co-labeling for immature neuronal marker, beta-III tubulin (TuJ). TuJ labeling reveals penetration of efferents into the developing adrenal gland (AG) and staining in the suprarenal sympathetic ganglion (SRG). With rare exceptions (arrows), there is almost no overlap in cells or processes expressing these respective markers. The kidney is identified (ki). B) The E14.5 AG exhibits consolidation of the TuJ staining into fibers that localize to the site of a7G labeled cell accumulation. Rare cells are labeled by both a7G and TuJ (black filled arrow). Note the coincident staining between these markers in the DRGs, but only very rare TuJ identified filaments in the kidney. C) Increased magnification of the E14.5 adrenal gland shown in B. In these images the cell exhibiting both a7G and TuJ labeling is identified by the black arrow. Aggregated a7G labeled cells with the presence of extensive TuJ-labeled afferents is identified for two of these clusters by arrows. Also present are a7G-labeled cells with no apparent association with TuJ labeled afferents (arrow heads). In some instances the TuJ identified afferents form fine fibers with varicosities that appear to wrap a7G stained cells (black filled arrow with asterisk). D) Shown are images from the AG medulla at the developmental stage indicated double labeled for a7G or neurofilament-M (NF-M). Arrows identify cells expressing a7G that appear to also be in association with NF-M stained afferents. Note the consolidation and apparent trimming of NF-M afferents and their association with clusters of a7G cells. E) Sections of the AG at the indicated developmental time are double labeled with a7G and the neurotrophin receptor subunit p75. Although no co-labeling of cells or processes with a7G and p75 was identified, there is extensive association between a7G cells and p75 labeled afferents that persists into the post-natal period (P13 shown) and becomes refined to include almost preferentially cell clusters of the adrenal medulla identified by the a7G-labeled cell clusters in the adult (arrows). This is particularly evident in the adjacent panels showing these clusters at increased magnification. There are however, some cell clusters in the adult that exhibit weak or no a7G immunostaining that are innervated by p75 stained fibers (asterisk). Occasionally p75-labeled processes also extend into the adrenal cortex (open arrow). These processes are not detected by anti-GFP staining. F) The adult immunostaining by the neurofilament protein-H (NF-H) exhibits less specificity for afferents interacting with a7G stained cells when compared to those afferents identified by p75. Abbreviations: AG, adrenal gland; ctx, adrenal cortex; ki, kidney; med, adrenal medulla; SRG, suprarenal ganglion. Scale bar = 100 mm (A,B, E adult) or 50 mm. doi:10.1371/journal.pone.0103861.g005

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Gahring, L. C., Myers, E., Palumbos, S., & Rogers, S. W. (2014). Nicotinic receptor alpha7 expression during mouse adrenal gland development. PLoS ONE, 9(8). https://doi.org/10.1371/journal.pone.0103861

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