The EGFR is required for proper innervation to the skin

Abstract

EGFR family members are essential for proper peripheral nervous system development. A role for EGFR itself in peripheral nervous system development in vivo, however, has not been reported. We investigated whether EGFR is required for cutaneous innervation using Egfr null and skin-targeted Egfr mutant mice. Neuronal markers; including PGP9.5, GAP-43, acetylated tubulin, and neurofilaments; revealed that Egfr null dorsal skin was hyperinnervated with a disorganized pattern of innervation. In addition, receptor subtypes such as lanceolate endings were disorganized and immature. To determine whether the hyperinnervation phenotype resulted from a target-derived effect of loss of EGFR, mice lacking EGFR expression in the cutaneous epithelium were examined. These mice retained other aspects of the cutaneous Egfr null phenotype but exhibited normal innervation. The sensory deficits in Egfr null dorsal skin were not associated with any abnormality in the morphology or density of dorsal root ganglion (DRG) neurons or Schwann cells. However, explant and dissociated cell cultures of DRG revealed more extensive branching in Egfr null cultures. These data demonstrate that EGFR is required for proper cutaneous innervation during development and suggest that it limits axonal outgrowth and branching in a DRG-autonomous manner.

Figure 1. Ablation of Egfr disturbs cutaneous innervation after E17.5

(a–d) GAP-43 immunofluorescence (green) shown in E17.5 skin. (a) Photomicrograph of skin section showing normal organization of sensory nerves of the skin in the wild type. Note that the normal pattern of nerve plexuses has emerged at this age; the deep dermal plexus (double-headed arrows), and radial fibers (large arrows). (b) Photomicrograph of skin section of the Egfr null. Note the lack of difference between innervation pattern and density between the wild-type skin (a) and the mutant. (c) A higher magnification image of skin section showing the deep dermal plexus (double-headed arrows), the radial bundles (large arrows), subepidermal plexus (small arrows), and epidermal free nerve endings (arrowheads). (d) The deep dermal plexus (double-headed arrows), the radial fibers (large arrows), subepidermal plexus (small arrows), and epidermal free nerve endings in Egfr null skin in a pattern and density comparable to the wild-type skins in (c). Scale bar is 100 μm (a–d). (e) The density of innervation is increased the skin of Egfr null mice beginning at P0. Nerve fiber density was quantified in PGP9.5- immunostained dorsal skin sections as described in the Materials and Methods. The relative area of PGP9.5-positive fibers was determined in 30 microscopic fields for each of three mice in each group. The mean for each mouse was calculated and the average area of innervation for each genotype at several time points is shown. Asterisk (*) indicates the mean is significantly different in the Egfr null skin compared to the corresponding wild-type control, where P≤0.05.

Figure 2. Sensory innervation is disorganized in P0 dorsal skin from Egfr null mice

(a) Photomicrographs of cutaneous sensory nerves in control mouse skin, labeled with PGP9.5, showing normal organization of sensory nerves into deep dermal plexus (double-headed arrows), radial fibers (large arrows), subepidermal plexus (small arrows), and epidermal free nerve endings (arrowheads). (b) Cutaneous innervation of Egfr null skin. Note the increased density and absence of normal pattern. The normal plexiform arrangement was replaced by a rete of nerves throughout the superficial layer of the dermis. (c) Acetylated tubulin immunolabeled sensory nerves of the wild-type skin showing the normal pattern of deep dermal plexus (double-headed arrows), subepidermal plexus (small arrows), and epidermal free nerve branches (arrowheads). (d) Acetylated tubulin immunolabeling of the cutaneous nerves of Egfr null showing consistently denser and disorganized pattern of sensory fibers. Note that epidermal and follicular keratinocytes (green cells) are immunostained for keratin 14 (K14) in (c) and (d). (e) Normal innervation pattern of the wild-type skin as revealed by GAP-43 immunolabeling. (f) Cutaneous innervation of Egfr null showing denser and disorganized pattern with GAP-43 immunolabeling. Scale bar is 100 μm in all figures.

Figure 3. Increased and disorganized sensory innervation in P5 Egfr null dorsal skin

(a, c, e) Acetylated tubulin, PGP9.5, and GAP-43 immunolabeled sensory nerves of the wild-type skin. The deep dermal plexus (double-headed arrows), and the subepidermal plexus (small arrows) are parallel to the skin surface. The radial fibers (large arrows) are obliquely oriented and parallel to the hair follicles, and are located at regularly spaced intervals. The lanceolate endings (circles) have started to form around several hair follicles. (b, d, f) Sensory nerves in the Egfr null skin. The normal three horizontal plexuses are replaced by single thick horizontal plexus (double-headed arrow) in the mid dermis, from which extensive set of radial fibers freely interlacing with massive secondary terminals. Note the extensive branching of the free nerve ending in the epidermis arrowheads in (d). Scale bar is 100 μm in all figures.

Figure 4. Egfr null skin exhibits immature and disorganized lanceolate endings

(a) Neurofilament-labeled normal, mature lanceolate ending in wild-type P7 skin, with its circular fibers (arrows) and vertical palisade (arrowheads). (b) A malformed lanceolate ending in P7 Egfr null skin (arrows). Note the absence of palisade and aberrant circular fibers. (c) Neurofilament staining of a mature lanceolate ending (arrows) in P18 control skin. (d) Malformed lanceolate ending in P18 Egfr null skin (arrows). Scale bar is 100 μm in all figures.

Figure 5. Ablation of Egfr in the epithelium of the skin results in disorganized hair follicles but normal innervation

(a) Immunoblotting of skin-targeted Egfr mutant (Egfr^fl/fl/Cre⁺) epidermis demonstrates loss of EGFR protein when compared to control (Egfr^fl/fl/Cre). Bar graph indicates relative EGFR signal normalized to actin from densitometry of immunoblots (N = 14 mice). Hematoxylin and eosin-stained skin sections from 7- to 8-day-old Egfr null (b) and skin-targeted Egfr mutant mice (c) exhibit disorganized hair follicles when compared to the normal control (d). PGP9.5-labeled (e–h) and neurofilament-labeled (i, j) nerve fibers of 14 day-old skin-targeted mutant dorsal skin (f, h, j) show normal innervation. Note that the deep dermal plexus, subepidermal plexus, radial fibers, and lanceolate endings are comparable to those in the wild type at 14 day (e, g, i). Scale bar is 100 μm in all figures.

Figure 6. Analysis of Egfr null and wild-type DRG reveals no differences

(a, b) Neurofilament 200-immunostained neurons of the wild-type and Egfr null Mid-DRG section at P5. Note the absence of any morphological difference or any difference in the density of neurons. (c, d) S-100-immunostained Schwann cells of wild-type and null Mid-DRG sections at P5, showing size and density similarity in both genotypes. (e, f) Toluidine-blue-stained wild-type and null P5 DRG sections for cell counting. Cells were quantified as described in the Materials and Methods and numbers indicate mean DRG cells±SEM. N = 3 mice. No significant difference between genotypes was detected using a Student’s t-test.

Figure 7. DRG explants and dissociated cells display aberrant branching upon ablation of Egfr

(a) Photomicrograph of wild-type P0 whole DRG explant after 4 days in culture, and labeling with PGP9.5. (b) Egfr null P0 DRG explant displays more branches than the wild-type littermate. Wild-type (c) and Egfr null (d) P0 dissociated DRG neurons labeled with PGP9.5. The Egfr null neurons demonstrate significantly increased branching compared to the littermate controls. Numbers indicate mean branch points ±SEM quantified as described in the Materials and Methods. Mean for the Egfr null is significantly greater than for the controls using a Student’s t-test, where P≤0.0001. N = 30 cells. Scale bar is 1 mm.