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First published online 24 July 2008
doi: 10.1242/dev.023960

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Abnormal sympathetic nervous system development and physiological dysautonomia in Egr3-deficient mice

¹ Department of Pathology, Northwestern University, Chicago, IL 60611, USA.
² Department of Neurology, Northwestern University, Chicago, IL 60611, USA.
³ Division of Neuropathology, Northwestern University, Chicago, IL 60611, USA.

View larger version (48K): [in this window] [in a new window]   Fig. 1. Egr3 expression is developmentally regulated and coupled to NGF signaling in SCG neurons. (A) At E13, Egr3 expression was low in SCG neurons and, by E15, Egr3 expression was markedly upregulated when sympathetic neurons begin to express TrkA and to respond to NGF. Tyrosine hydroxylase (TH) expression confirmed the representation of sympathetic neurons in all of the RNA samples (results from five to eight pooled ganglia for each developmental time point and qPCR performed in triplicate, *P<0.001; n.s., no significant difference, Student's t-test relative to E13 time point). (B) Egr3 expression was not detectable at E13 by in situ hybridization, whereas at (C) birth (P0) it was expressed by most, if not all SCG neurons. cs, carotid sinus; ca, carotid artery; scg, superior cervical ganglion; scale bars: 100 µm. (D) Treatment with NGF function-neutralizing antibody resulted in a 60% reduction in Egr3 expression relative treatment with PBS, indicating that Egr3 expression was coupled to NGF signaling in SCG neurons in vivo (results from n=5 PBS and NGF treated P0 mice and qPCR performed in triplicate; **P<0.01, Mann-Whitney U test). (E) Egr3 expression was induced sixfold by NGF treatment, but not after treatment with the neurotrophins NT4 or BDNF. NGF-dependent Egr3 induction was abrogated using the MEK inhibitor U0126 (results from three experimental replicates and qPCR performed in triplicate; *P<0.001, Student's t-test relative to control).

View larger version (117K): [in this window] [in a new window]   Fig. 2. Widespread sympathetic neuron loss without migration defects in postnatal Egr3-deficient mice. (A) Sympathetic neuron loss was detected in the SCG from Egr3-/- mice starting 1 day after birth (P1). Approximately 30% of SCG neurons were lost between P0 and P1, and the difference between wild-type and Egr3-/- mice persisted in aged adult mice when the overall numbers declined because of physiological attrition of senescent neurons (results from three or four SCG per genotype and developmental time point; *P<0.05, Student's t-test relative to wild type). (B-E) At P1, the sympathetic ganglia were properly positioned along the rostral-caudal axis. (B) The SCG was smaller (black arrowhead) and many axon bundles emanating from it were either absent (broken outline) or markedly atrophic (white arrowhead) in Egr3-/- mice. Similarly, (C) the stellate ganglion (STG; black arrowhead), (D) the thoracic paravertebral chain ganglia and (E) caudal sympathetic ganglia were consistently smaller (black arrowheads), and there was atrophy and loss of projections emanating from them (white arrowheads) (representative results from three P1 Egr3+/+:DlZ and Egr3-/-:DlZ mice; scale bars: 250 µm).

View larger version (54K): [in this window] [in a new window]   Fig. 3. Increased apoptosis in sympathetic neurons in Egr3-/- mice. (A) No significant differences in apoptosis were observed until P0, at which point apoptosis in Egr3-/- SCG was nearly double that of wild type (results of Casp3+ cells from two to six ganglia of each genotype and developmental time point, *P<0.01, Student's t-test). Representative images of ganglia of wild-type (right, top) and Egr3-/- mice (right, bottom) (results from cultures performed in triplicate). Scale bars: 40 µm. (B) Cellular proliferation (BrdU+ cells) was not altered in the SCG between wild-type and Egr3-/- mice at P0. (C) There was no autonomous defect in NGF-dependent survival of Egr3-/- SCG neurons in vitro. There was a highly significant correlation between neuron survival and NGF concentration, but no significant difference in the amount of survival between wild-type and Egr3-/- neurons at any NGF concentration tested. Representative images of sympathetic neurons stained with Hoechst/DAPI (right, top) and Casp3 (right, bottom) (results from cultures performed in triplicate).

View larger version (116K): [in this window] [in a new window]   Fig. 4. Decreased sympathetic innervation to target organs is accompanied by abnormalities in axon extension and terminal axon branching in Egr3-/- mice. (A) In the submandibular gland and sublingual gland (broken outline) from Egr3+/+:DlZ+ mice, lacZ histochemistry revealed robust sympathetic innervation. (B) Framed area in A: sympathetic axons branched into the distal lobules of the glands (arrowheads). (A') In Egr3-/-:DlZ+ glands, there was a relative decrease in sympathetic innervation, consistent with sympathetic neuron loss. (B') Framed area in A': there was less complex axon branching and numerous axons that failed to extend to the distal lobules of the glands (arrowheads). (C) In trachea from Egr3+/+:DlZ+ mice, sympathetic innervation entered along the dorsal midline and branched circumferentially to innervate smooth muscle and submucosal glands (arrowheads). (C') In trachea from Egr3-/-:DlZ+ mice, however, sympathetic axon branching was consistently decreased and the branching of remaining axons was markedly diminished. (D,D') Sympathetic axons entered the splenic parenchyma along the splenic arteries (black arrowhead) and Egr3+/+:DlZ+ spleens (D) the axons branched extensively after entering the organ parenchyma (white arrowheads). By contrast, in Egr3-/-:DlZ+ mice (D'), sympathetic axons branched poorly as they entered the parenchyma (white arrowheads). Global sympathetic innervation to the spleen could be observed in Egr3+/+:DlZ+ spleens (D, right), which showed a diffuse blue reaction product (arrow) that was consistently diminished in Egr3-/-:DlZ+ spleens (D', right) (arrows). (Representative results from three adult mice for each genotype and organ analyzed; scale bars: 250 µm.)

View larger version (85K): [in this window] [in a new window]   Fig. 5. Physiological blepharoptosis in Egr3-/- mice. Compared with wild-type mice (A), Egr3-/- mice (A') have prominent blephoroptosis (drooping eyelids). (B) Whole-mount lacZ histochemistry of the inner eyelids from adult Egr3+/+:DlZ+ mice showed dense sympathetic innervation to the superior and inferior tarsal muscles (black arrowheads) and Meibomian glands in the inner eyelids (columnar innervation, white arrowheads). (B') Sympathetic innervation was markedly decreased to the upper and lower eyelids (black arrowheads) and the Meibomian glands (white arrowheads) in Egr3-/-:DlZ+mice. Compared with wild type (C), adult Egr3-/- mice (C') developed corneal ulceration (arrow), most probably owing to impaired secretomotor function of the Meibomian glands. Scale bars: 0.5 mm. *nasal canthus.

View larger version (75K): [in this window] [in a new window]   Fig. 6. Impaired sympathetic innervation and altered AANAT induction in pineal glands from Egr3-/- mice. The pineal glands from (A) wild-type and (A') Egr3-/- mice appeared histologically indistinguishable. Immunohistochemistry for TH showed robust sympathetic innervation to the (B) wild-type pineal glands that was either highly diminished or absent in most pineal glands from Egr3-/- mice (B'). Similar results were obtained with the lacZ sympathetic reporter that showed a robust network of sympathetic axons in (C) wild-type pineal glands that was barely detectable in (C') pineal glands from most Egr3-/- mice. (D) AANAT is induced by increased sympathetic activity to the pineal upon entry into the dark phase of the light-dark cycle. In 10D:14L light cycle entrained wild-type mice, AANAT expression was induced as expected during the dark phase of the light-dark cycle and returned to low basal levels when sympathetic activity to the pineal gland decreased with the onset of the light phase of the cycle. However, in Egr3-/- pineal glands AANAT was induced to 18% of the wild-type level, consistent with a physiological impairment of sympathetic innervation to the gland. The white and black bar along the horizontal axis depicts the time during the light-dark cycle when the lights are on and off, respectively. Zeitgeber time (ZT) represents the 24 hour elapsed time in the light-dark cycle relative to the time of dark-light transition (ZT0). (AANAT expression representative of three independent experiments and qPCR performed in triplicate; the peak wild-type value at ZT21 was considered 100% maximal AANAT induction in this paradigm; scale bars: 25 µm.)

View larger version (83K): [in this window] [in a new window]   Fig. 7. Abnormal cardiac sympathetic innervation and autonomic dysfunction in Egr3-/- mice. Whole-mount lacZ histochemistry was performed on hearts from adult Egr3+/+:DlZ+ and Egr3-/-:DlZ+ mice. In Egr3+/+:DlZ+ mice, sympathetic innervation to the inferior surface of the (A) right atrium and the ventral surfaces of the (B) right and (C) left ventricles could be visualized in detail. (C) Diffuse innervation of the myocardium was visualized in Egr3+/+:DlZ+ hearts (arrowhead). (A'-C') In hearts from Egr3-/-:DlZ+ mice, however, there was a marked decrease in the overall epicardial sympathetic innervation and the small terminal axon branching was markedly diminished (results are representative of three Egr3+/+:DlZ+ and Egr3-/-:DlZ+ hearts analyzed by whole-mount lacZ histochemistry). (D,E) Decreased sympathetic innervation to the heart was accompanied by abnormal physiological response to sympathetic nervous system activation. (D) Heart rate and (E) contractility measurements were similar between untreated (baseline) wild-type and Egr3-/- mice. Treatment with the central 2-adrenergic antagonist yohimbine (YOH) resulted in a greater than twofold increase in heart rate and myocardial contractility in wild-type mice relative to baseline owing to increased sympathetic activity to the heart. In Egr3-/- mice treated with YOH, the increase in heart rate and myocardial contractility was significantly diminished compared with wild-type mice (results from five wild-type and seven Egr3-/- catherized mice; *P<0.01, Student's t-test; scale bars: 1 mm in A,B; 0.5 mm in C).

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