Topic Article
The EGFR Is Required for Proper Innervation to the Skin
Adel Maklad, Jodi R Nicolai, Kyle J Bichsel, Jackie E Evenson, Tang-Cheng Lee, David W Threadgill and Laura A Hansen
Journal of Investigative Dermatology (2009) 129. 690-698; doi: 10.1038/jid.2008.281

Epidermal Growth Factor Receptor Regulates Skin Nerve Outgrowth and Branching
Robb Marchione ¹, Nancy Kim ¹ and Robert S. Kirsner ¹
Journal of Investigative Dermatology (2009) 129, 524. doi:10.1038/jid.2008.462

Among its many important functions, skin serves as an interface with the environment, including providing the ability to sense pain, touch, vibration, and temperature. To accomplish this, the skin is richly innervated, with complex interplay between skin cells and the neural system in mediating cutaneous sensation. This is supported by a highly organized pattern of three horizontal nerve plexuses (Botchkarev et al., 1997; Maklad et al., 2004). The epidermal growth factor receptor (EGFR) family appears to be important in nervous system development (Casalini et al., 2004). This family of proteins includes EGFR, Erbb2/HER2, Erbb3/HER3, and Erbb4/HER4, all of which are expressed in the nervous system (Casalini et al., 2004). Some members of this family—Erbb2, Erbb3, and Erbb4—have been strongly implicated in neural development (Lee et al., 1995; Lin et al., 2000), but EGFR has not yet been well studied.

To begin to understand the role of EGFR in neural development in the skin, Maklad and co-workers (2009) utilized egfr-null mice, which lack EGFR receptors entirely, and mutant mice, which lack EGFR receptors in the skin. When these mice were compared with wild-type mice, differences were observed after initial embryonic development of the nervous system but during times when a peripheral nervous system “pruning” effect would normally occur. Overall, the egfr-null mice had a hyperinnervated but disorganized array of cutaneous nerves; this pattern was also observed with receptor subtypes such as lanceolate endings. Using skin-specific mutant EGFR it was found that EGFR is required for development of the sensory component of the peripheral nervous system contributed by the dorsal root ganglion (DRG), but not DRG or Schwann cell development. In sum, EGFR appears to be important in regulating axonal outgrowth and branching, independent of the DRG.
Through the following questions, we examine this paper in greater detail.

QUESTIONS
1. How do growth factors and growth factor receptors work?
2. What is the rationale for studying EGFR in the nervous system?
3. How did the investigators test their hypotheses?
4. What were the major findings of the study?
5. What may be the clinical implications of the study?
6. What studies could be performed to confirm or further the observations reported in this article?

ANSWERS

1. The foundation of a cell’s ability to communicate with its surrounding environment rests on receptor–ligand interactions that take place at the cell surface. There are innumerable such interactions—driving cellular function, growth, differentiation, migration, and apoptosis and coordinating these processes on a macrobiological scale. Herein we consider the interaction between epidermal growth factor (EGF) and its corresponding receptor complex.

The EGF receptor (EGFR) is a member of the receptor tyrosine kinase (RTK) family, which includes many of the primary mediators of cellular growth and differentiation. RTK activity is modulated via the allosteric binding of a number of known and unknown extracellular ligands. This ligand binding induces either homo- or heterodimerization of RTK at the cell surface, requisite for the tyrosine kinase autophosphorylation activity that initiates a cascade of downstream events. Activation of RTK at the cell surface precipitates a distinct transcriptional program that includes mitogen-activated protein kinase (MAPK); phosphatidylinositol-3 kinase (PI-3K); the proto-oncogenes ras, fos, jun, and myc; and transcription factors such as Sp1, Egr1, CREB, and members of the Ets family of transcription factors. In Homo sapiens, the human EGFR (HER) family consists of four receptors: HER-1 (also called EGFR or erbB-1), HER-2 (also called erbB-2 or Neu), HER-3 (also called erbB-3), and HER-4 (also called erbB-4) (Casalini et al., 2004).

The numerous possible receptor–ligand interactions characteristic of the HER family explain the great diversity of potential downstream messages inherent in this system. HER ligands can be divided into three groups that exhibit different receptor-binding specificities while all sharing an EGF-like motif of 45–55 amino acids. The first group, which binds specifically to HER-1, includes EGF, amphiregulin, and transforming growth factor-α (TGF-α). The second group, which binds to HER-1 and HER-4, includes betacellulin and heparin-binding EGF. The third group can be further divided into two subgroups based on whether the ligands bind HER-3 and HER-4 or HER-4 only, and this group includes the neuregulins (Casalini et al., 2004).

At a minimum, there are nine different homo- and heterodimers of HER. The many ligands that can bind to EGFRs have different abilities with respect to activation of unique receptor dimers. In turn, the various receptor dimers have distinct autophosphorylization sites that are responsible for the recruitment of different combinations of downstream effector molecules (Casalini et al., 2004). With this basic framework, one can begin to appreciate how this population of growth factors and growth-factor receptors works to initiate action at a cellular level.

2. At first blush it is not surprising that the skin and nervous system share many signaling molecules. Both systems derive from the ectoderm, and the densely innervated skin serves as the nervous system’s interface with the external environment. There is a body of evidence supporting a role for EGF and EGFR in multiple facets of nervous system development. Each of the four member receptors is expressed in the nervous system, and studies have implicated the EGFR family in nervous system development. It has been known for more than a decade that mice lacking EGFR develop both epithelial and neurological disease, resulting in death within 1 month of birth (Sibilia and Wagner, 1995). Cellular localization studies have indicated that EGFR appears early in rat cerebellar and cortex development, supporting a role for EGF in central nervous system (CNS) development (Wong and Guillaud, 2004). EGF stimulates neurite outgrowth, in addition to increasing dopamine uptake and enhancing long-term survival of cultured dopaminergic neurons (Yamada et al., 1997). TGF-α, an EGFR ligand, is expressed in proliferating cells of rat basal ganglia, the germline zone, and the ventricular zone of the medial ganglionic eminence. Furthermore, EGFR is constitutively expressed in regions undergoing active neurogenesis, including the subventricular zone, the granule layer of the dentate gyrus, and the cerebellar granule layer in the postnatal rat, with attenuation of this expression to the subventricular zone in the adult rat (Cameron et al., 1998). Upon activation of the EGFR pathway, astrocytes in the developing mouse nervous system differentiate to form the cribiform structure that surrounds axons (Liu and Neufeld, 2007). In vitro studies of cultured astrocytes have suggested that EGF acts as a mitogen along with inducing increased glutamine synthetase activities in these cells (Yamada et al., 1997).

As Maklad et al. (2009) indicate, a number of null mice models have revealed a role for the various members of the EGFR family in the development of the nervous system. Erbb2 null mice display a complete lack of cutaneous innervation whereas the sensory neurons of the cranial ganglia in Erbb4 null mice have deficient pathfinding ability. Furthermore, mice with an HER-3 null mutation, Cre-targeted HER-2 ablation in Schwann cells, or mutations in the ligand, neuregulin-1, all exhibit anomalous motorneuron axons that defasciculate as they enter the muscle mass and fail to form mature neuromuscular junctions.

Taken together, these data suggest that the moniker “epidermal growth factor” is misleading. This growth factor/receptor system clearly has a role that extends deeper than the epidermis and maintains a prominent role in various aspects of nervous system function and development.

3. Given the abundance of data supporting a role for EGFR in CNS development, the investigators sought to explore the receptor’s potential role in peripheral nervous system (PNS) development using in vivo models. In particular, the investigators wanted to study the role of EGFR in the development of cutaneous innervation. They employed two genetic models, one with the Egfr gene knocked out of the entire mouse genome and another model that selectively knocked out the gene at the level of the mouse epidermis only. The null mice (Egfr ⁺) were created using conventional methods that are similar to those described in the answers to the February 2009 Journal Club. To create the selective knockout mice, the investigators placed loxP sites flanking an exon in the Egfr gene, then crossed these mice with transgenic mice expressing a Cre recombinase gene driven by a keratin promoter, guaranteeing selective expression in the epithelial cells of the epidermis. When Cre recombinase is expressed, it clips out sequences flanked by loxP sites. Immunoblotting of Cre (–) and Cre (+) mice was performed to guarantee selective disruption of EGFR expression in the epidermis. In this model, the Cre recombinase selectively disrupts Egfr expression in the epidermis only and provides insight into this gene’s role solely at the level of the skin (LA Hansen, personal communication).

The investigators sought to characterize the effect that eliminating Egfr expression, both from the entire genome and at the level of the epidermis, would have on the development of cutaneous sensory innervation. The entire sensory circuit was examined, from the effects on distal cutaneous innervation to the more central dorsal root ganglia (DRG). To determine whether the effect of EGFR ablation was specific to the fields innervated by the DRG, the authors compared the innervation patterns in the mouse foot-pad skin with that of whisker-pad skin in Egfr null mice and their wild-type littermates. Whisker-pad skin represented cutaneous targets supplied by trigeminal ganglia. To determine whether the ablation of EGFR had any effect on autonomic nervous system development, tyrosine hydroxylase (TH) immunofluorescence was conducted. TH is involved in first-step catecholamine synthesis by converting tyrosine to dopa, and is more specific for this class of neurons than other immunofluorescence methods.

Dorsal skin biopsies from Egfr null mice, Egfr-selective knockout mice, and wild-type littermates were compared. The investigators examined biopsies from progressively younger mice to determine the time at which a difference between mutants and wild-type controls emerged, determining grossly when EGFR starts to function in PNS development. Immunolabeling using GAP-43, PGP9.5, acetylated tubulin, and neurofilament antibodies was used to effectively demonstrate the deep dermal plexi, subepidermal plexi, radial fibers, and lanceolate endings.

To determine whether the anomalous cutaneous hyperinnervation occurring in Egfr null mice was secondary to increased DRG perikarya, the investigators evaluated DRG sections from null and wild-type mice stained with neurofilament 200, S-100, and toluidine blue. After seeing no difference between null and wild-type DRG sections, they cultured Egfr DRG neurons to test whether ablation of Egfr increases neurite branching and outgrowth.

Taken together, these methods were an attempt to shed light on the role of EGFR in the development of peripheral sensory innervation. By selectively ablating EGFR expression from the final target of the DRG, the investigators began to define the receptor’s specific site of action.

4. Comparison between dorsal skin innervation of Egfr null mice and their wild-type control littermates. Overall, EGFR was found to attenuate the density of cutaneous innervation. Beginning at embryonic day 17.5 (E17.5), there was no difference in the organization or quantity of cutaneous innervation between Egfr null and wild-type mice, as shown by growth-associated protein-43 (GAP-43) antibodies and PGP9.5 immunolabeling, respectively. Immunolabeling using GAP-43, PGP9.5, and acetylated tubulin antibodies showed disorganized cutaneous hyperinnervation at postnatal day 0 in Egfr mice. Interestingly, the terminal branches were more abundant in Egfr null mice. A progressively greater disparity in cutaneous innervation was noted between Egfr null and wild-type mice through postnatal day 18. This disparity was characterized by denser free nerve terminals in the epidermis, defective lanceolate-ending development, and disorganized connections among the three major cutaneous nerve plexi. Lastly, the aberrant cutaneous innervation of Egfr null mice was confined to fields originating with the DRG, not the trigeminal ganglia, and did not seem to involve the peripheral autonomic nervous system.

Skin-targeted Egfr mutant mice have normal cutaneous sensory innervation. As described above, a population of mice lacking EGFR expression in only the epidermis was created. Since Egfr null mice exhibited severely disorganized hair follicles, the investigators attempted to determine whether this phenotype was responsible for the aberrant cutaneous innervation observed in these mice. Similarly to the Egfr null mice, the skin-targeted mutants exhibited disorganized hair follicles. However, the organization and density of all cutaneous nerve plexi exhibited a normal pattern indistinguishable from that of wild-type skin. These data indicated that neither epithelial EGFR expression nor normal hair follicle organization is required for normal peripheral cutaneous sensory innervation.

In vivo examination of Egfr null DRG cell populations reveals no significant difference from wild-type correlates, and in vitro Egfr null DRG cell cultures exhibit increased neurite branching. Since no innervation defects were observed in skin-targeted Egfr mutants, the investigators sought to determine whether disorganized cutaneous hyperinnervation originated in the DRG. Neurofilament 200 and S-100 markers were used to examine nerve cells and Schwann cells. Toluidine blue–stained neuronal cells were also observed. These studies revealed no differences in cell morphology or density between the Egfr null and wild-type populations, suggesting that the Egfr null phenotype results from increased and disorganized neurite branching originating in the periphery.

To determine whether EGFR ablation increases neurite branching in vitro, Maklad et al. (2009) cultured whole DRG explants and dissociated DRG cells from Egfr null mice. Manual counting of the branch points of axonal arbors revealed 60% more branch points in the Egfr null populations. This data, together with the DRG histology data described above, suggests that EGFR is involved in attenuating neurite branching during PNS development at the level of growing, path-finding neurons.

5. The moniker “epidermal growth factor receptor” is misleading. EGFR may serve a role in epidermal cell growth and differentiation, but it has other functions as well, not the least of which is a role in pruning the advancing sensory neurons during PNS development. This role in PNS development suggests a possible clinical application. In particular, a role for the inhibition of EGFR in optic nerve regeneration has been touted as an exciting source of potential therapies (Berry et al., 2008). Axon regeneration in the adult CNS is limited by inhibitory molecules associated with myelin and the glial scar. It has been shown that suppression of EGFR kinase activity blocks both myelin inhibitors and chondroitin sulfate proteoglycans from inhibiting neurite outgrowth. Furthermore, the administration of local EFGR inhibitors promotes the regeneration of optic nerve fibers (Koprivica et al., 2005). The EGF/EGFR system’s role of neurite paring may also be applied to the CNS. Perhaps EGFR inhibitors can be employed to encourage neurite branching in hopes of reestablishing connections in injured axons of the CNS.

6. These investigators have demonstrated effectively that EGFR is essential for normal PNS development and that the receptor functions in the regulation of PNS pattern formation and the inhibition of nerve branching. By ablating Egfr expression in the epidermis, they showed that expression of this receptor in these cells is not required for normal nerve plexus development. Thus, the expression of EGFR in the target of sensory neuron migration is not required for a normal phenotype. However, there are numerous cell types with which the developing neurons come into contact during their migration to the skin surface. Granted, in vitro data argue against the role of EGFR expression in other cell types directing neuronal development. However, future experiments may be designed that selectively knock out Egfr expression from dermal cells such as fibroblasts in order to determine how the milieu of the developing neuron contributes to its development, as the in vitro data are not conclusive.

The role of EGFR in neuron development appears to function in a cell-autonomous manner; that is, Egfr-null DRGs exhibited normal morphology despite a severely disorganized cutaneous innervation pattern. To ascertain whether the cell autonomous model is correct, DRG-targeted Egfr mutant mice could be created and the elegant immunolabeling experiments repeated.

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¹ Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA