RA decreases <i>EDN3</i> and <i>Ece1</i> mRNA levels, depending on whether exogenous EDN3 is present.

<p>p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Relative <i>EDN3</i> (A-B) and <i>Ece1</i> (C-D) mRNA levels were measured by quantitative RT-PCR after 3 (A, C) and 14 (B, D) days. Levels are normalized to β-actin and the -RA/-EDN3 condition was standardized to a value of 1. Solid lines denote a statistically significant difference (p<0.05) between “No RA” and “RA”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”. (A) At 3 days, RA was associated with a net decrease in the level of <i>EDN3</i> in the absence and presence of exogenous EDN3 (C) Similarly, RA was associated with a decrease in the relative abundance of <i>Ece1</i>, albeit only in the presence of EDN3 After 14 days, RA still decreased the relative abundance of <i>EDN3</i> (B) and <i>Ece1</i> (D) mRNA.</p

Effects of RA and EDN3 on the terminal culture morphology of ENS precursors.

<p>p75^NTR immunoselected cells were grown for 14 days in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Culture morphology was evaluated by immunofluorescence microscopy. Peripherin expression (red) is consistent with neuronal morphology and myofibroblasts are indicated by expression of α-SMA (green). The third and fourth columns include DAPI staining for nuclei. The first three columns were photographed at 10x (Scale bar = 100 µm) and the fourth column was photographed at 40x (Scale bar = 50 µm). In the absence of EDN3 and RA (EDN3-RA-), SMA+ myofibroblasts predominated, but sparse neurons were also seen. EDN3+RA- treated cultures formed a homogeneous sheet of SMA+ myofibroblasts. In EDN3-RA+ cultures, neurons formed a plexus punctuated by large multicellular ganglia, abundant peripherin+ neurites and cell bodies, and non-myofibroblast-like SMA+ cells. SMA+ myofibroblasts were also present beneath the plexus. With EDN3+RA+ treatment, many peripherin+ neurons and long complex neurites were seen, without forming a plexus. Myofibroblasts, while excluded from neuronal regions, were still present in large number.</p

RA increases S100β+ and decreases peripherin+ cell prevalence while EDN3 decreases S100β+ cell prevalence.

<p>p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. The proportion (A and C) and proliferating fraction (B and D) of peripherin- and S100β-immunoreactive cells were quantified. Solid lines denote a statistically significant difference (p<0.05) between “No RA” and “RA”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”. (A) The proportion of S100β+ cells was enriched by RA and decreased in response to EDN3 (B) RA increased, and EDN3 decreased the fraction of proliferating S100β+ cells. (C) the proportion of peripherin+ cells decreased in response to RA treatment, but EDN3 did not have an effect. (D) Neither RA nor EDN3 had an effect on proliferation of peripherin+ cells.</p

A hypothetical model depicting the bidirectional relationship of RA and EDN3 signaling in ENS precursors.

<p>The above pictogram, based on our data, summarizes and integrates the observed RNA expression profiles and the culture morphological data. Solid green arrows indicate increases in mRNA levels and solid red arrows with flat arrowheads denote decreases in mRNA levels. Although RA and EDN3 levels were not quantified in our culture model, dashed green and red arrows denote putative RA synthesis and degradation, respectively. We hypothesize that RA inhibits EDN signaling in ENS precursors by modulating the gene expression of <i>EDN3</i> and <i>Ece1</i>, and EDN3 regulates RA signaling by inhibiting RA receptor expression, thereby decreasing RA responsiveness, and fine-tuning RA metabolic enzyme expression, putatively altering RA availability. We propose that RA perpetuates glia by decreasing <i>Ret</i> and enhancing <i>Sox10</i> expression, and that EDN3 prevents the proliferation of glia by decreasing <i>Sox10</i>. The persistence of glia is conducive to the formation of a heterocellular ganglionated plexus. This model suggests that control of <i>Sox10</i> marks a key developmental decision point - a switch - that sustains multipotent progenitors in culture and ultimately depends on the balance of RA and EDN3</p

Culture in the presence of RA increases <i>Sox10</i>, but EDN3 suppresses this effect.

<p>p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Relative <i>Sox10</i> (A–B) and <i>Ret</i> (C–D) mRNA levels were measured by quantitative RT-PCR after 3 (A, C) and 14 (B, D) days. Expression is normalized to β-actin and the -RA/-EDN3 condition was standardized to a value of 1. Solid lines denote a statistically significant difference (p<0.05) between “No RA” and “RA”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”. (A) In the absence of EDN3, RA increased <i>Sox10</i> levels; however, the concurrent addition of EDN3 abolished this phenomenon. There was a statistically significant interaction (ⓧ; p=0.019) between EDN3 and RA in their effect on <i>Sox10</i> levels at 3 days. This interaction is specific to <i>Sox10</i> expression and was not observed with <i>Ret</i>. (B) RA treatment was associated with increased <i>Sox10</i> levels but EDN3 had no effect at 14 days. (C) RA decreased <i>Ret</i> levels at 3 days regardless of whether EDN3 was present, but EDN3 did not affect <i>Ret</i> gene expression. (D) Decreased <i>Ret</i> levels in response to RA persisted at 14 days, but only in the presence of EDN3</p

Effects of RA and EDN3 on the terminal culture morphology of ENS precursors.

<p>p75^NTR immunoselected cells were grown for 14 days in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Culture morphology was evaluated by immunofluorescence microscopy. Peripherin expression (red) is consistent with neuronal morphology and glia are indicated by expression of S100β (green). The third and fourth columns include DAPI staining for nuclei. The first three columns were photographed at 10x (Scale bar = 100 µm) and the fourth column was photographed at 40x (Scale bar = 50 µm). Without EDN3 and RA (EDN3-RA-) sparse neurons developed, but S100β+ glia were more numerous and more brightly staining. EDN3+RA- cultures did not contain glia or neurons. EDN3-RA+ cultures formed discrete heterocellular ganglia with abundant peripherin+ cell bodies and S100β+ glia, linked by thick peripherin+ neurites in a plexus pattern. EDN3+RA+ treatment also contained many peripherin+ neurons and long complex neurites, but these neurons were disorganized, and did not form a plexus. Weakly staining S100β+ cells with glial cell morphology were also found amidst the neurons. The glia in these cultures were more fusiform and were mainly associated with neurons.</p