Egr3 Dependent Sympathetic Target Tissue Innervation in the Absence of Neuron Death

Nerve Growth Factor (NGF) is a target tissue derived neurotrophin required for normal sympathetic neuron survival and target tissue innervation. NGF signaling regulates gene expression in sympathetic neurons, which in turn mediates critical aspects of neuron survival, axon extension and terminal axon branching during sympathetic nervous system (SNS) development. Egr3 is a transcription factor regulated by NGF signaling in sympathetic neurons that is essential for normal SNS development. Germline Egr3-deficient mice have physiologic dysautonomia characterized by apoptotic sympathetic neuron death and abnormal innervation to many target tissues. The extent to which sympathetic innervation abnormalities in the absence of Egr3 is caused by altered innervation or by neuron death during development is unknown. Using Bax-deficient mice to abrogate apoptotic sympathetic neuron death in vivo, we show that Egr3 has an essential role in target tissue innervation in the absence of neuron death. Sympathetic target tissue innervation is abnormal in many target tissues in the absence of neuron death, and like NGF, Egr3 also appears to effect target tissue innervation heterogeneously. In some tissues, such as heart, spleen, bowel, kidney, pineal gland and the eye, Egr3 is essential for normal innervation, whereas in other tissues such as lung, stomach, pancreas and liver, Egr3 appears to have little role in innervation. Moreover, in salivary glands and heart, two tissues where Egr3 has an essential role in sympathetic innervation, NGF and NT-3 are expressed normally in the absence of Egr3 indicating that abnormal target tissue innervation is not due to deregulation of these neurotrophins in target tissues. Taken together, these results clearly demonstrate a role for Egr3 in mediating sympathetic target tissue innervation that is independent of neuron survival or neurotrophin deregulation

Target tissue innervation abnormalities in the absence of sympathetic neuron death in Egr3-deficient mice.

<p>(<b>A</b>) In the absence of the pro-apoptotic molecule Bax, sympathetic neurons in the SCG were protected from both physiologic and Egr3-dependent death. Approximately 1/3 of SCG neurons were lost in Egr3-deficient (3-B+) compared to wild type (3+B+) mice. In the absence of Bax, physiologic sympathetic neuron death (3+B−) and Egr3-dependent neuron death (3-B−) was inhibited. Inhibition of physiologic death increased the total number of SCG neurons as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025696#pone.0025696-Glebova1" target="_blank">[10]</a>. (number of SCG analyzed from 8–12 week old mice indicated in parentheses; *  =  p<0.01 and n.s. =  non-significant compared to 3+B+ mice, Student's t test) (<b>B</b>) In the absence of Egr3 and inhibited apoptosis (3-B−), SCG neurons were significantly atrophic. (8–12 week old mice analyzed, *  =  p<0.001 compared to 3+B−, Student's t test and size-frequency, χ²<0.0001). (<b>C</b>) Similar qualitative results were observed in thoracic prevertebral ganglia as apoptotic death was rescued in all sympathetic neurons. Compared to wild type ganglia (3+B+), 3-B+ ganglia were notably smaller and ganglia lacking Bax, with or without loss of Egr3 were larger. All mice were 8-12 weeks of age and expressed the DτlZ sympathetic reporter transgene to highlight the ganglia after LacZ histochemical staining (scale bar  =  25 µm).</p

Abnormal sympathetic innervation to small bowel in the absence of Egr3 and sympathetic neuron death.

<p>(<b>A</b>) in wild type (3+B+) mice, tyrosine hydroxylase (TH) immunohistochemistry performed on vascularized intact small bowel demonstrated sympathetic axons coursing along the mesenteric blood vessels (arrowhead) and prominent intercrossing pattern of TH+ fibers in the bowel wall (arrow). (<b>B, C</b>) In preparations from mice lacking Egr3, with (3-B+) our without Bax (3-B−), the TH+ axons were noted to prematurely defasciculate from the mesenteric blood vessels prior to innervating the bowel wall (arrowhead) and the pattern of intercrossing TH+ fibers in the bowel wall was highly attenuated (arrow) (4 week old mice shown). (<b>D, E</b>) The attenuated mural innervation was confirmed using DτlZ sympathetic reporter mice which showed a highly attenuated innervation of the bowel wall in 3-B− relative to 3+B− mice. (<b>F</b>) Sympathetic innervation to the small bowel mucosa was observed in the vascular core of mucosal villi from control (3+B−; DτlZ+) sympathetic reporter mice (arrow, villi shown in whole mount preparation in the axial plane). (<b>F</b>') Sympathetic innervation within the small bowel villi paralleled the vascular capillary network (villus shown in whole mount preparation in the longitudinal plane). (<b>G</b>) In Egr3-deficient mice with inhibited sympathetic neuron death (3-B−; DτlZ+) <45% of the villi were innervated (arrow) and (<b>G</b>') most villi that did have some innervation showed a highly attenuated abnormal innervation pattern. (quantitative results represent the mean ± standard error of >500 villi scored; * = p<0.01, Student's t test compared to 3+B−; DτlZ+ control mice; scale bar = 50 µm; number of 4–8 week old animals analyzed indicated in parentheses).</p

Renal sympathetic innervation abnormalities in the absence of Egr3 and sympathetic neuron death.

<p>(<b>A, B</b>) Lacz histochemistry highlighted diffuse renal cortical sympathetic innervation in mice expressing the DτlZ reporter transgene. (<b>B</b>) In the absence of Egr3 and sympathetic neuron death (3-B−;DτlZ) there was a marked decrease in lacZ staining in the glomerular zone (gz) of the renal cortex compared to (<b>A</b>) control (3+B−; DτlZ) mice. (scale bar = 500 µm) (<b>C, D</b>) The decreased sympathetic innervation in the gz was correlated with a 40% reduction of (<b>C, D</b>) sympathetic innervation (TH immunofluorescence, left) to the renin expressing juxtaglomerular (jg) apparatus (renin immunofluorescence, middle) within the gz. (scale bar = 50 µm) (results of TH immunofluorescence quantification representing the mean ± standard error in the area juxtaposed to the jg apparatus; * = p<0.01, Student's t test compared to 3+B−; DτlZ+ control mice; number of 4–8 week old animals analyzed indicated in parentheses).</p

Cardiac sympathetic innervation abnormalities in the absence of Egr3 and sympathetic neuron death.

<p>Sympathetic innervation to the (<b>A</b>) right and (<b>B</b>) left ventricles of the heart was reduced by 58% and 65%, respectively in (<b>A</b>'<b>, B</b>'') 3-B− mice compared to (<b>A, B</b>) 3+B− mice. (scale bar = 50 µm) (results of TH immunofluorescence quantification representing the mean ± standard error; * = p<0.01, Student's t test compared to 3+B− mice; number of 4–8 week old animals analyzed indicated in parentheses).</p

Disruption of sympathetic target tissue innervation originating from the SCG in the absence of neuron death in Egr3-deficient mice.

<p>(<b>A</b>) Compared to 8 week old Bax-deficient control mice (3+B−) (<b>B</b>) physiologic ptosis and (<b>B</b>') innervation abnormalities to the eye were present in the absence of Egr3 and Bax (3-B−). Results show lacZ staining in mice also expressing the DτlZ sympathetic reporter transgene which highlighted (<b>B</b>') superior tarsus muscle innervation (arrowheads) and meibomian gland (arrows) innervation abnormalities to the eyes in 3-B− mice compared to (<b>A</b>') 3+B− control mice. (* = nasal canthus; scale bar = 100 µm). (<b>C, C</b>') Sympathetic innervation to the pineal gland, a major target of SCG neurons, was reduced by >50% in 3-B− mice compared to 3+B− control mice (4–8 weeks old). (scale bar = 200 µm)</p

Target tissue innervation in 3-B− mice compared to 3+B− mice.

<p>+  =  highly reduced innervation relative to adult 3+B− control mice.</p><p>+++ and ++  =  intermediate innervation relative to adult 3+B− control mice.</p><p>++++  =  similar innervation relative to adult 3+B− control mice.</p><p>p values, Student's t test, from semi-quantitative TH immunofluorescence.</p

Normal sympathetic innervation was observed in some tissues in the absence of Egr3 and neuron death.

<p>In brown fat for example, which receives dense sympathetic innervation to regulate thermogenesis no definitive changes were observed in (<b>A</b>) 3+B− relative to (<b>B</b>) 3-B− mice. (scale bar  = 100 µm).</p