The Human Mechanosensory Corpuscles: A New Schwann Cell Localization of the Wilms’ Tumor Protein WT1

Abstract

Summary

The Wilms’ Tumor protein WT1 is a zinc-finger transcription factor with crucial roles in organogenesis, cell differentiation, tissue homeostasis, and oncogenesis. While its expression has been extensively studied in various tissues, its presence in the nervous system, particularly in peripheral glial cells, remains largely unexplored. In this study, we examined WT1 expression in the Schwann cells of mechanosensory corpuscles, nerve bundles, and free nerve endings (FNEs) within human penile tissues. Using single and double immunohistology, we analyzed WT1 coexpression with Schwann cell markers (S100, nestin, SOX10) and its association with axonal (neurofilaments, neuron-specific enolase, tyrosine hydroxylase) and perineurial/endoneurial markers (Glut-1, α-SMA, CD34). We found consistent WT1 cytoplasmic expression in the Schwann cells of Pacinian, Meissner, Krause, genital, Golgi-Mazzoni, and Ruffini-like corpuscles, with variable staining intensity. Confocal microscopy revealed WT1 colocalized with nestin but not S100, suggesting involvement in cytoskeletal organization. In addition, we documented WT1 in myelinating Schwann cells of nerve bundles, with distinct staining patterns in Cajal bands and Schmidt–Lanterman incisures, as well as in non-myelinating Schwann cells of FNEs. This is the first study to describe WT1 expression in sensory corpuscles, implicating it in Schwann cell development, maintenance, or plasticity, with potential relevance for peripheral nerve biology, pathology, and mechanosensation.

Introduction

The non-neuronal inner core cells of mechanosensory corpuscles are recognized as specialized, modified non-myelinating Schwann cells (nmSCs) of neural crest origin, commonly referred to as lamellar cells or terminal glial cells.^1,2 In 1881, Krause ³ proposed that the inner core cells of sensory corpuscles originate from Schwann cells, a concept largely supported by subsequent literature through ultrastructural, ² histochemical, ⁴ immunohistochemical, ⁵ experimental denervation,^1,4 and transplantation studies.^6,7 Among peripheral glial subtypes, these cells exhibit distinct morphological and functional properties, including their complex cytoplasmic lamellar organization around axonal endings ⁸ and involvement in mechanotransduction,^{9 –12} which distinguish them from other Schwann cell populations in the peripheral nervous system. ¹³ Based on the lamellar pattern of inner core Schwann cell cytoplasm, corpuscular receptors can be classified into three groups: ⁴ those with asymmetrical lamellation (Meissner, Krause, genital, and Golgi-Mazzoni corpuscles), those with symmetrical lamellation (Pacinian and Paciniform corpuscles), and those lacking a defined lamellar arrangement (Ruffini corpuscles).

In Meissner corpuscles, located within dermal papillae, the modified Schwann cells are typically arranged parallel to the epithelial surface, forming a distinctive coin-stack configuration with peripherally positioned nuclei. ¹⁴ In contrast, Krause corpuscles, genital corpuscles, and Golgi-Mazzoni corpuscles, typically located deeper in the dermis, contain Schwann cells with an irregular organization, which in genital corpuscles often form lobular structures partitioned by CD34⁺ endoneurial septa, a common immunohistological characteristic of their architecture.^15,16 In Pacinian corpuscles, as Schwann cells lose their myelin sheaths in the preterminal zone of the inner core, they adopt a bilaterally symmetric arrangement in the terminal zone. ² This zone consists of two regular hemilamellar systems that terminate opposite each other at the so-called radial clefts, which are dual gaps positioned 180° apart. However, this symmetry is lost in the ultraterminal zone, where it disintegrates into multiple lamellar systems, each associated with individual axonal processes. Limited data are available in the published literature regarding the morphological organization of Schwann cells in Ruffini corpuscles. ¹⁷ However, Chouchkov ⁴ reported that Schwann cell cytoplasmic lamellae do not accumulate around the Ruffini axonal endings.

In addition to providing structural support and directly participating in mechanotransduction, corpuscular Schwann cells are also proposed to play roles in regulating extracellular ionic homeostasis (K⁺, Na⁺, Cl⁺, and Ca²⁺), ⁸ providing neurotrophic support, and transmitting mechanical forces to axonal terminals via adherens junctions, ¹⁸ which are essential for subsequent Piezo2-mediated axonal activation. Furthermore, they are thought to fine-tune axonal depolarizations through the release of gamma-aminobutyric acid, glutamate, and potentially other signaling molecules,^19,20 while also adopting a repair phenotype in response to injury or denervation.^4,21 Despite these proposed functions, corpuscular Schwann cells remain one of the least understood and least studied subtypes of peripheral glia. Recent reviews by Hastings and Valdez ²¹ and Suazo et al. ⁸ summarize the available structural and functional data on these complex yet poorly characterized glial cells.

Adult Schwann cells within sensory corpuscles express a distinct set of molecular markers that can be categorized into several groups.^5,8 These include cytoskeletal intermediate filaments (vimentin, nestin, and occasionally glial fibrillary acidic protein GFAP), calcium-binding proteins (S100, parvalbumin, calbindin), growth factors and their receptors (BDNF, NGFR, TrkA, TrkB, EGFR), cell cycle proteins (Bcl-2), transcription factors (SOX10), cell adhesion molecules (CD56/NCAM), and ion channels involved in mechanosensitivity, or mechanoproteins (TRPV4, ASIC2). This classification is based on human data and immunohistological analyses,^5,8 but additional molecules have been identified in corpuscular Schwann cells of non-human species, including the transmembrane protein Ush2A in mouse forepaw Meissner corpuscles ²² and the metabotropic glutamate receptor 1 (Grm1) in cat mesenteric Pacinian corpuscles. ²⁰ SOX10, a marker of the Schwann cell lineage expressed throughout all stages of Schwann cell development, stands out among these markers as the only one that selectively immunostains the nuclei of corpuscular Schwann cells, as demonstrated in our previous study on the human prepuce. ¹⁵ However, existing data likely capture only a small subset of potential immunoreactivities in corpuscular Schwann cells, highlighting the need for further research to map the full molecular landscape governing their differentiation, maintenance, and function.

WT1

The Wilms’ Tumor protein WT1 is a zinc-finger transcription factor primarily involved in tissue and organ development, ²³ sex determination, ²⁴ adult tissue homeostasis, ²⁵ and tumorigenesis.^26,27 The WT1 gene, located on chromosome 11p13, encodes at least 24 protein isoforms in humans, regulating target genes involved in cell differentiation, growth, proliferation, and apoptosis. Furthermore, WT1 proteins exert multifaceted effects through transcriptional and post-transcriptional regulation, mRNA splicing, and protein–protein interactions. ²³ In adult tissues, a dramatic example of WT1’s vital functions in tissue homeostasis was provided by Chau et al., ²⁵ who demonstrated that ubiquitous deletion of WT1 in adult mice led to rapid multiple organ failure, including glomerulosclerosis, pancreatic atrophy, bone and fat loss, and failure of erythropoiesis, highlighting WT1’s critical role in maintaining mesenchymal and hematopoietic stem cell lineages . In recent years, WT1 has emerged as a key player in the central and peripheral nervous systems, contributing to the development of the visual, ²⁸ olfactory, ²⁹ and gustatory sensory systems, ³⁰ the central regulation of locomotion,^31,32 synaptic plasticity and memory flexibility, ³³ central respiratory control, ³⁴ and neuronal degeneration in Alzheimer’s disease. ³⁵ These findings underscore WT1’s broad regulatory influence in the nervous system, inviting further exploration of its potential roles beyond the central nervous system, particularly in peripheral nerve biology and glial function.

In immunohistochemistry, two commonly used WT1 antibodies target distinct regions of the protein, influencing their immunostaining patterns in both normal and pathological tissues. The polyclonal antibody C-19 recognizes multiple epitopes within the C-terminal region, resulting in nuclear staining, consistent with WT1’s role as a transcription factor. ³⁶ In contrast, the monoclonal antibody 6F-H2 targets the N-terminal region, producing both nuclear and cytoplasmic staining, ³⁷ reflecting WT1’s ability to shuttle between the nucleus and cytoplasm, a property linked to its diverse functions. ³⁸ WT1 cytoplasmic staining was previously regarded as spurious or a result of cross-reactivity with an unrelated epitope but is now recognized as a normal aspect of WT1 immunolocalization.^37,39 This differential localization suggests WT1’s involvement in transcriptional regulation, mRNA processing, and cytoplasmic functions, emphasizing the importance of antibody selection when interpreting WT1 expression in tissue sections.

To the best of our knowledge, WT1 expression in sensory corpuscles has not been explored in the published literature, although WT1 has been identified in human normal peripheral neural tissue using immunohistochemistry. Schittenhelm et al. ⁴⁰ reported WT1⁺ Schwann cells ensheathing myelinated axons within nerve bundles in normal adult peripheral nerves, while the axons themselves lacked WT1 immunoreactivity. They described two distinct WT1 cytoplasmic staining patterns: in transversely sectioned axons, WT1 exhibited a punctate or dot-like immunoreactivity in the Schwann cells surrounding the axons (refer to their Fig. 1D), which they attributed to partial staining of the Schwann cells. In longitudinally sectioned axons, WT1 staining intensity varied along the length of the axon, with some areas appearing completely surrounded by WT1⁺ Schwann cells (refer to their Fig. 1E). Subsequently, Inagaki et al. ⁴¹ corroborated the presence of WT1 in the Schwann cell cytoplasm of normal adult peripheral myelinated nerves, specifically in cauda equina nerve roots (see their Fig. 1C), and additionally noted WT1 expression within the myelin sheaths. The accounts of WT1⁺ normal myelinating Schwann cells (mSCs) by Schittenhelm et al. ⁴⁰ and Inagaki et al. ⁴¹ were limited and lacked detailed analysis. Later, Parenti et al. ⁴² demonstrated WT1⁺ Schwann cells in human fetal peripheral nerves, providing a developmental explanation for the findings in adult tissues reported by Schittenhelm et al. ⁴⁰ and Inagaki et al. ⁴¹

Figure 1.

Preputial Pacinian corpuscles with WT1 expression in the inner core Schwann cells, demonstrated through single and double immunohistochemistry. (A, B) Serially sectioned Pacinian showing weak WT1 immunoreactivity without linker in A and stronger WT1 expression with signal amplification via linker in the consecutive section (B). WT1 signal amplification was applied in all subsequent WT1 microphotographs in this study unless otherwise specified. (C) Pacinian corpuscle showing strong WT1 staining in the inner core. Note the adjacent WT1⁺ endothelial staining (arrows). (D, E) Serially sectioned Pacinians: (D) NF⁺ punctate axonal profiles in the inner cores (brown) and WT1⁺ inner core Schwann cells (magenta); (E) WT1 in the inner cores (brown) and Glut-1 in the outer cores (magenta). (F) Pacinian corpuscle with WT1⁺ inner core (brown) and SOX10⁺ nuclear immunoreactivity (magenta) in inner core Schwann cells. Some SOX10⁺ nuclei are obscured due to chromogen overlap with strong WT1 staining, though a clearly visible SOX10⁺ nucleus is present in the peripheral inner core. (G, H) Serially sectioned small Pacinian corpuscle: (G) WT1⁺ lamellar cells in the inner core (brown) with NF⁺ axonal profiles (magenta). An additional axonal profile is visible in the lower portion of the image, within the peripheral part of the outer core; (H) WT1⁺ lamellar cells in the inner core (brown) and CD34⁺ cells in the intermediate layer. CD34⁺ fibroblast-like cells or dermal dendrocytes (magenta) surround the Pacinian. (I) Pacinian corpuscles with WT1 expression in the inner cores (brown) and CD34 in the intermediate layers (magenta). (J–L) Pacinian corpuscles showing WT1 expression in the inner core (magenta) and SMA immunoreactivity in the capsule (brown). Surrounding the corpuscles are small smooth muscle bundles and blood vessels. In the upper left corner of J, note a blood vessel with WT1⁺ endothelium (magenta) and an SMA⁺ tunica media (brown). Adjacent SMA⁺ smooth muscle bundles and WT1⁺ endothelial cells are also visible. In L note multiple WT1⁺ Schwann lamellar cell systems within the enlarged Pacinian inner core (magenta), potentially representing a transitional form between the Pacinian and Golgi-Mazzoni corpuscles. Scale bars = 100 µm (A–F, I, J–L), 50 µm (G, H).

The characteristic dot-like or punctate expression pattern of WT1⁺ mSCs documented by Schittenhelm et al. ⁴⁰ resembles the typical immunostaining pattern of the so-called Cajal bands in peripheral nerves observed in cross-section.^43,44 Similarly, the WT1⁺ mSCs described by Inagaki et al., ⁴¹ associated with longitudinally sectioned axons, resemble the immunostaining pattern of Cajal bands when observed in axonal longitudinal sections. ⁴³ Cajal bands are longitudinal and transverse mesh-like abaxonal cytoplasmic channels within internodal Schwann cells, ⁴⁵ believed to play a role in nerve fiber development and demyelinating neuropathies such as Charcot–Marie–Tooth disease. ⁴⁶ The cytoplasmic channels are compartmentalized by adjacent appositions formed by the outer surface of the myelin sheath and the cytoplasmic side of the Schwann cell plasma membrane, containing periaxin–dystrophin-related protein 2 (DRP2-dystroglycan complex).^45,47 They were first described by Ramón y Cajal ⁴⁸ around 1914 in teased preparations of myelinated sciatic nerve fibers from adult cats. He postulated that these cytoplasmic bands might play a role in nutrient transport within Schwann cells. In 2004, Court et al. ⁴⁹ renamed them “Cajal bands” and proposed that they play a role in Schwann cell growth, facilitating microtubule-mediated centrifugal transport of mRNA from the nucleus to distal sites for local translation, a mechanism similar to that observed in oligodendrocytes. Given that WT1 expression in normal mature Schwann cells has only been seemingly reported in two isolated studies,^40,41 with brief descriptions and no in-depth analysis, it is unsurprising that no studies have yet proposed hypotheses or provided insights into its potential roles in these specialized neuroglial cells.

Recent in-house, unpublished analyses from our laboratory, using light microscopic immunohistochemistry, demonstrated WT1 cytoplasmic expression in the Schwann or lamellar cells of all types of human penile preputial sensory corpuscles. The penile prepuce has long been recognized for its rich and diverse array of specialized corpuscular receptors,^{50 –59} which contribute to the normal complement of penile erogenous sensation.^53,60 These include Meissner corpuscles, Pacinian corpuscles, genital corpuscles, Krause corpuscles, Ruffini corpuscles, and mucocutaneous corpuscles. ⁵⁹ These findings prompted further investigation to evaluate WT1 expression patterns and precise cellular localization within normal penile neural structures.

Thus, this study aimed to provide the first comprehensive characterization of WT1 immunohistological expression in the human mechanosensory corpuscles using adult penile tissues. In addition, we examined WT1 expression in nerve bundles and free nerve endings (FNEs) of the human prepuce, a tissue also characterized by a dense plexus of nerve bundles and a high concentration of FNEs.^57,61 To further investigate the molecular profiles of WT1⁺ neural structures, we performed single and double immunohistochemistry on selected sections, using markers for endoneurium (CD34), perineurium (α-SMA, Glut-1), Schwann cells (nestin, S100, SOX10), and axons [neurofilaments, neuron-specific enolase (NSE), tyrosine hydroxylase (TH)] in various combinations. Furthermore, double immunofluorescence was performed to evaluate the potential colocalization of WT1 with nestin and S100 in Schwann cells.

Materials and Methods

This study complied with Spanish regulations and the ethical principles outlined in the 1964 Helsinki Declaration and its subsequent revisions. Prior approval was obtained from the Santiago-Lugo Research Ethics Committee (code 2021/179).

Post-circumcision Preputial Specimens

Twelve fresh preputial specimens were obtained from the Urology Service of the University Clinical Hospital of Santiago de Compostela, with written informed consent from all patients. The mean patient age was 26 years (range: 18–35 years, SD: 5.5). Circumcision was performed for phimosis or recurrent inflammation. Histopathological examination confirmed that all specimens were normal, although some displayed mild to moderate focal subepithelial inflammation. The specimens were fixed in 10% neutral-buffered formalin for 24 hr.

Cadaveric Specimens

Six intact (not circumcised) adult cadaveric penile specimens were also included in this study, all donated to the Body Donation and Dissecting Room Center of the Complutense University of Madrid for educational and research purposes. The mean donor age at the time of death was 66 years (range: 51–96 years, SD: 16). All specimens were obtained from fresh cadavers and subsequently immersion-fixed in 10% neutral-buffered formalin for several days following grossing to ensure adequate fixative penetration. In all cases, fixation occurred within 24 hr postmortem. The causes of death were unrelated to the genitourinary system, and no penile pathologies or history of penile surgery were identified, as confirmed by gross inspection. Each penile specimen included the prepuce, glans, midshaft, proximal penis, and penile root (including crura and bulb). Transverse sections of these penile regions were obtained from all specimens, along with separate fragments of preputial tissue.

Single and Double Immunohistochemistry

All tissue samples were dissected, placed in standard cassettes or Supa Mega (CellPath, Newton, Powys, UK; EAO-0102-02A) cassettes, and processed overnight using an automated tissue processor before being embedded in paraffin. Four-μm-thick serial and nearby sections from each tissue block were mounted on FLEX IHC microscope slides (Dako-Agilent, Glostrup, Denmark). Sections from each specimen were stained with hematoxylin and eosin (H&E) using a Dako-Agilent CoverStainer for basic histological and histopathological analysis. Immunostaining was carried out using the Autostainer Link 48 (Dako-Agilent) and the EnVision FLEX detection system (Dako-Agilent), following the manufacturer’s guidelines. Before staining, pretreatment was performed in a PT Link module (Dako-Agilent) for 20 min. The protocols requiring overnight incubation of the primary antibody (TH only) were carried out manually up to the incubation step and then completed using the Autostainer Link 48. Tissue antigens were visualized using EnVision FLEX/HRP diaminobenzidine and EnVision FLEX/HRP magenta chromogen solutions (Dako-Agilent). Sections were counterstained with hematoxylin. For each specimen and each automated immunohistochemical round, negative controls were processed without primary antibodies, while endothelial WT1 immunostaining served as an internal positive control. The complete step-by-step single- and double-staining immunohistochemical protocols are provided in the Appendix.

Antibodies

In addition to WT1, this study incorporated a panel of immunohistochemical markers to further characterize the molecular profile of neural structures where WT1 was detected. The antibodies and immunohistochemical protocols employed are detailed in Table 1. Specifically, α-SMA was used for smooth muscle and perineurial structures, Glut-1 specifically for perineurial structures, CD34 for endoneurial components, NF and NSE as general axonal markers, S100 and nestin for cytoplasmic staining of Schwann cells, SOX10 for selective nuclear Schwann cell staining, and TH for unmyelinated noradrenergic axons. More detailed information on the normal and pathological tissue antigens targeted by these markers can be found in the works by Ortiz-Hidalgo and Weller, ⁶² Reina et al., ⁶³ and Chetty et al. ³⁹

Table 1.

List of Antibodies and Corresponding Incubation Protocols Used in This Study.

Antibody (Clone)	Origin	Supplier	Code	Concentration	HIER pH	Time/Temperature
α-SMA (1A4)	Mouse	Dako-Agilent b	IR611	Ready-to-use	High pH	20 min/RT
CD34 (QBEnd 10)	Mouse	Dako-Agilent b	IR632	Ready-to-use	High pH	30 min/RT
Glut-1 (SPM498)	Mouse	Biocare Medical c	CM 408 A, B	1:100	High pH	30 min/RT
Nestin (EPR1301(2))	Rabbit	Abcam d	Ab176571	1:000	Low pH	20 min/RT
NF (2F11)	Mouse	Dako-Agilent b	IR607	Ready-to-use	High pH	20 min/RT
NSE (BBS/NC/VI-H14) a	Mouse	Dako-Agilent b	IR612	Ready-to-use	High pH	30 min/RT
S100 (polyclonal)	Rabbit	Dako-Agilent b	IR504	Ready-to-use	High pH	30 min/RT
SOX10 (BC34) a	Mouse	Biocare Medical c	ACI3099C	1:50	High pH	30 min/RT
TH (E2L6M) a	Rabbit	Cell Signaling Technology e	58844	1:50	High pH	24 h/4°C
WT1 (6F-H2) a	Mouse	Dako-Agilent b	IR055	Ready-to-use	High pH	20 min/RT

Abbreviations: α-SMA, alpha smooth muscle actin; CD, cluster of differentiation; Glut-1, glucose transporter 1; NF, neurofilament; NSE, neuron-specific enolase; SOX10, SRY (sex-determining region Y)-box 10; TH, tyrosine hydroxylase; HIER, heat-induced epitope retrieval; RT, room temperature; WT1, Wilms’ Tumor 1.

a

When necessary, an additional signal amplification step was performed for these antibodies using a 15-min incubation with the EnVision FLEX+ Mouse/Rabbit Linker (Dako-Agilent).

b

Glostrup, Denmark.

c

Pacheco, CA, USA.

d

Cambridge, UK.

e

Beverly, MA, USA.

Double Immunofluorescence Labeling

Double immunofluorescence was performed on selected slides to assess the possible colocalization of WT1 with nestin and S100 in Schwann cells of sensory corpuscles and nerve bundles, with visualization using confocal microscopy. The antibodies and antigen retrieval methods are detailed in Table 1. Briefly, WT1 mouse monoclonal antibody (Dako-Agilent, ready-to-use) was incubated for 24 hr at 4C, followed by a 1-hr incubation at room temperature (RT) with sheep anti-mouse IgG F(ab’)2 fragment conjugated with Cy3 (Sigma-Aldrich, St. Louis, MO) at a 1:200 dilution. Next, slides were incubated with either nestin rabbit monoclonal antibody (Abcam, clone EPR1301(2), Cambridge, UK, 1:1000 dilution) or S100 rabbit polyclonal antibody (Dako-Agilent, ready-to-use) for 24 hr at 4C. This was followed by a 1-hr RT incubation with Alexa 488-labeled donkey anti-rabbit IgG (Invitrogen, Life Technologies Corp, Eugene, OR) at a 1:200 dilution. Finally, nuclei were counterstained with DAPI nuclear stain (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 62248) at a 1:1000 dilution for 10 min at RT, in some but not all sections.

Digital Slide Scanning and Microphotograph Acquisition

Selected slides were fully digitized in brightfield mode at ×20 magnification using a PathScan Combi scanner (Excilone, Elancourt, France). Representative microphotographs were captured with PathScan software version 3.0.10. In addition, slides were light microscopically examined and photographed using an Olympus BX51 microscope (Tokyo, Japan) equipped with a digital camera (Olympus DP70, Tokyo, Japan).

For confocal microscopy image acquisition, visualization was performed using a Leica Stellaris 8 FALCON inverted laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with the Leica Application Suite X (LAS X) software package. Imaging was conducted with HC PL APO CS2 20x/0.75 Dry, 63x/1.30 Gly, and 93x/1.30 Gly objectives. Samples stained with DAPI, Alexa 488, and Cy3 were excited using a white light laser (WLL2) with excitation/emission wavelengths set at 405 nm/430–490 nm for DAPI, 498 nm/504–550 nm for Alexa 488, and 554 nm/560–625 nm for Cy3. Image stacks (xyz) were acquired in sequential mode with optimized xy and xz resolution. To further enhance resolution, Leica Lightning adaptive deconvolution was applied.

Results

WT1 Immunohistochemical Staining in Sensory Corpuscles

Preputial (Fig. 1), glans (Fig. 2), and bulbar (Fig. 3) Pacinian corpuscles consistently exhibited WT1 immunopositivity in their inner core Schwann lamellar cells. The staining intensity ranged from weak to moderate (typically moderate) without signal amplification, and from moderate to strong (typically strong) with signal amplification (Figs. 1A and B and 2A and B). Similarly, WT1 expression was observed in the lamellar or Schwann cells of preputial Golgi-Mazzoni (Fig. 4), Meissner (Fig. 5), Krause (Fig. 6A, B, D to F, and O), genital (Fig. 6C, G to J, M, and N), and Ruffini-like corpuscles (Fig. 6K and L). However, signal amplification was often required for optimal detection of WT1 specifically in Meissner corpuscles. In all sensory corpuscle types, the WT1 immunostaining pattern in the corpuscular Schwann cells was exclusively cytoplasmic, with clear nuclear sparing.

Figure 2.

Glans Pacinian and Paciniform corpuscles with WT1 expression in the inner core Schwann cells, demonstrated through single and double immunohistochemistry. (A–C) Serially sectioned small Pacinian corpuscle with double inner core and a common perineurial outer core: (A) Faint WT1 immunoreactivity in the inner cores without signal amplification; (B) strong WT1 expression in the inner cores following signal amplification; (C) WT1 in the inner cores (brown) and strong Glut-1 expression in the common outer core (magenta). (D, E) Serially sectioned small Pacinian corpuscle: (D) WT1 expression in the inner core with signal amplification (brown) and Glut-1 in the outer core (magenta). Note adjacent WT1⁺ nerve bundles encased by Glut-1⁺ perineurium; (E) WT1 expression in the inner core without signal amplification (brown) and CD34 in the intermediate layer (magenta). (F) Paciniform corpuscle displaying WT1 in the inner core (brown) and CD34 in the intermediate layer (magenta). Scale bars = 50 µm (A–E), 25 µm (F).

Figure 3.

Bulbar Pacinian and Paciniform corpuscles with WT1 expression in the inner core Schwann cells and WT1 immunoreactivity in related structures, all observed without signal amplification. (A) Low-magnification image of the bulbar region of the corpus spongiosum, with the bulbar septum (BS) visible on the left. (B) High-magnification view of the outlined area in A, showing a small Pacinian corpuscle with WT1 expression in the inner core, located within a bulbar trabecula. (C) Low-magnification image of the bulbar region of the corpus spongiosum, again showing the bulbar septum (BS) labeled. (D) High-magnification view of the outlined area in C, depicting a Paciniform corpuscle with WT1⁺ Schwann cells in the inner core. (E) Bulbar urethral Littré glands exhibiting intense WT1⁺ staining. (F) Nerve bundle within the penile suspensory apparatus displaying WT1 immunoreactivity in the Cajal bands of Schwann cells (arrows), surrounded by adipose tissue. Scale bars = 400 µm (A, C), 100 µm (B, D), 50 µm (E, F).

Figure 4.

Preputial Golgi-Mazzoni corpuscles with WT1 expression in the inner core Schwann cells, demonstrated through single and double immunohistochemistry. (A, B) Serially sectioned Golgi-Mazzoni corpuscle: (A) WT1⁺ Schwann cells in the inner core (brown) and NF⁺ axons in the inner core (magenta); (B) WT1 expression in the inner core (brown) and CD34⁺ cells positioned between the inner and outer core, extending into the inner core (magenta). Unlike Pacinian corpuscles (Figs. 1H and I and 2E and F), a well-defined CD34⁺ intermediate layer is absent. (C) Golgi-Mazzoni corpuscle displaying numerous NF⁺ axonal profiles in the inner core (brown) and Glut-1 immunoreactivity in the outer core/capsule (magenta). The capsular layers appear loosely arranged, in contrast to the more compact capsules observed in the other Golgi-Mazzoni corpuscles in this figure. (D, E) Serially sectioned Golgi-Mazzoni: (D) WT1⁺ Schwann lamellar cells in the inner core (brown) with strong Glut-1 staining in the compact outer core (magenta); (E) WT1⁺ Schwann cells in the inner core (brown) and SOX10 nuclear staining in the same cells (magenta). (F) Consecutive serial section of the Golgi-Mazzoni corpuscle shown in C, exhibiting WT1⁺ cells in both the inner and outer core, an uncommon finding. (G, H) Serially sectioned Golgi-Mazzoni: (G) HE staining; (H) WT1 expression in the lobulated inner core (brown) and CD34⁺ fibroblast-like cells (possible telocytes) with long, slender processes extending between the inner and outer core and into the inner core (magenta), compartmentalizing the lamellar cells. As observed previously, a well-defined CD34⁺ intermediate layer, characteristic of Pacinian corpuscles, is absent. (I) Another typical Golgi-Mazzoni corpuscle, showing NF⁺ axonal profiles in the inner core (brown) and CD34⁺ cells (magenta) situated between the inner and outer core, as well as within the inner core itself, resembling the distribution patterns seen in B and H. Scale bars = 50 µm (A–I).

Figure 5.

Preputial papillary corpuscles with WT1 expression in Schwann cells, demonstrated through single and double immunohistochemistry. (A, B) Serially sectioned Meissner corpuscle: (A) WT1 immunoreactivity is prominent in the Schwann cell cytoplasm, with no staining in the nuclei; (B) consecutive section showing NF⁺ axons (magenta) transversely oriented relative to the corpuscle’s long axis. Note the absence of CD34⁺ capsule. (C) Papillary corpuscle displaying faint WT1 immunoreactivity without signal amplification (brown) and a dense network of glomerularly arranged NF⁺ axons (magenta). (D, E) Serially sectioned Meissner corpuscle: (D) WT1 strong expression is restricted to the Schwann cell cytoplasm, with unstained nuclei; (E) consecutive section showing transversely arranged NF⁺ axons (magenta) and no CD34⁺ capsule. (F) Papillary corpuscle with a dense NF⁺ axonal network (brown) and weak to strong WT1 immunoreactivity in the lamellar cells (magenta). A Schwann cell at the center of the corpuscle exhibits a strong perinuclear WT1⁺ immunostaining pattern (magenta). (G, H) Serially sectioned rounded Meissner corpuscle: (G) WT1 staining is confined to the Schwann cell cytoplasm, with no expression in the nuclei; (H) consecutive section showing NF⁺ axons and absent CD34⁺ capsule. (I) Meissner corpuscle with NF⁺ axons aligned transversely to its long axis and WT1⁺ Schwann cells displaying cytoplasmic immunoreactivity (magenta). (J, K) Serially sectioned Meissner corpuscle: (J) HE staining; (K) WT1⁺ Schwann cells (brown) with SOX10⁺ nuclear expression in the same cells (magenta); (L) rounded subepithelial corpuscle with dense glomerular NF⁺ axons (brown) and WT1⁺ Schwann cells (magenta). (M) Papillary corpuscle with WT1⁺ lamellar cells (brown) and CD34⁺ adjacent dermal fibroblast-like cells (magenta). No distinct capsule is observed. (N) Meissner corpuscle with densely packed NSE⁺ axons (brown) and faint WT1 staining in the lamellar cells (magenta). (O) Small Meissner corpuscle with NF⁺ axons (brown) enclosed by WT1⁺ lamellar cells showing strong immunoreactivity (magenta). Scale bars = 50 µm (A–O).

Figure 6.

Preputial deep dermal Krause, genital, and Ruffini-like corpuscles with WT1 expression in Schwann cells. (A, B) Serially sectioned encapsulated Krause corpuscle: (A) WT1⁺ Schwann cells within the corpuscle, with some capsular cells exhibiting perinuclear and cytoplasmic WT1 expression, an uncommon finding; (B) well-defined CD34⁺ capsule (brown) and NF⁺ axons (magenta) inside the corpuscle. (C) Large encapsulated genital corpuscle with WT1⁺ lamellar Schwann cells compartmentalized into lobules. (D) Small encapsulated Krause corpuscle displaying WT1⁺ Schwann cells (brown) and a CD34⁺ capsule (magenta). (E, F) Two small encapsulated Krause corpuscle with WT1⁺ Schwann cells (brown) and Glut-1⁺ capsules (magenta). (G, H) Serially sectioned genital corpuscle: (G) WT1⁺ Schwann cells in the inner core (brown) and a Glut-1⁺ capsule (magenta). The capsule exhibits Glut-1⁺ perineurial continuity with an adjacent small nerve bundle supplying the corpuscle. On the left, a small Pacinian or Paciniform corpuscle, likely sectioned through its Glut-1⁺ capsule (magenta), is visible, with its inner core out of the plane of section; (H) higher magnification of the outlined region in G, highlighting WT1⁺ Schwann cells (brown) and SOX10⁺ Schwann cell nuclei (magenta). (I) Large genital corpuscle with dense NF⁺ axonal profiles (brown) surrounded by WT1⁺ Schwann cell cytoplasm (magenta). (J) Large genital corpuscle with an SMA⁺ capsule (brown) and WT1⁺ lamellar Schwann cells organized in lobules (magenta). (K) Ruffini-like fusiform corpuscle displaying WT1⁺ Schwann cell cytoplasm (brown) and SOX10⁺ Schwann cell nuclei (magenta). (L) Ruffini-like fusiform corpuscle with dense NF⁺ axons (brown) enveloped by WT1⁺ Schwann cell cytoplasm (magenta). Note the axons supplying the corpuscle on the left part of the image, also associated with WT1⁺ Schwann cells (magenta). (M) Large genital corpuscle containing densely packed NF⁺ axons (brown), surrounded by WT1⁺ Schwann cell cytoplasm (magenta). (N) Pair of Krause or genital corpuscles showing WT1⁺ Schwann cells (brown) and NSE⁺ axonal profiles (magenta). (O) Probable Krause corpuscle featuring WT1⁺ Schwann cells (brown). No Glut-1⁺ perineurial capsule is observed. Scale bars = 50 µm (A–F, H–O), 100 µm (G).

Pacinian corpuscles consisted of an inner core containing axons and Schwann lamellar cells, an intermediate layer, an outer core, and a surrounding capsule, which was sometimes indistinct. Preputial Golgi-Mazzoni corpuscles exhibited a characteristic structure, with a large inner core containing axons and Schwann lamellar systems, an irregular intermediate layer extending projections into the inner core, and a relatively small outer core or capsule. Sensory corpuscles in the papillary dermis formed a highly variable group. Many were typical Meissner corpuscles, characterized by a long axis perpendicular to the epithelial surface, stacked Schwann lamellar cells aligned parallel to the epithelium with peripherally positioned nuclei, and axons ascending between the flattened lamellar cells. However, the papillary corpuscles also included receptors with a less structured inner core, where axons were glomerularly arranged between Schwann cells without a well-defined organization. These corpuscles were typically non-encapsulated or only partially encapsulated. Krause corpuscles appeared as small to medium-sized, encapsulated, rounded structures with glomerularly arranged axons, typically located deep in the dermis. Genital corpuscles were generally larger than Krause corpuscles, encapsulated, and often displayed internal Schwann cell lobulations along with dense axonal proliferations. Like Krause corpuscles, they were located in the deep dermis. Ruffini-like corpuscles were identified as fusiform structures located in the deep dermis.

Double immunohistochemistry was employed to characterize the molecular profiles of WT1⁺ Pacinian (Figs. 1D to L and 2C to F), Golgi-Mazzoni (Fig. 4A to E, H, and I), Meissner (Fig. 5C, F, I, and K to O), Krause (Fig. 6B, D to F, and O), genital (Fig. 6G to J, M, and N), and Ruffini-like corpuscles (Fig. 6K and L). Briefly, axons across all types of sensory corpuscles consistently exhibited NF and NSE immunoreactivity. Regardless of corpuscle type or anatomical location, intracorpuscular Schwann cells expressed S100, SOX10, and nestin. In both Pacinian and Golgi-Mazzoni corpuscles, the outer core was Glut-1⁺ (Figs. 1E, 2C and D and 4C and D) but only Pacinians exhibited a well-defined CD34⁺ intermediate layer (Figs. 1H and I and 2E and F). In contrast, the CD34⁺ intermediate layer of Golgi-Mazzoni corpuscles was less distinct and extended projections into the inner core (Fig. 4B, H, and I). Occasionally, a distinct α-SMA⁺ capsular layer encasing the Pacinian outer core was observed, occurring in approximately 10–20% of Pacinian corpuscles (Fig. 1J to L). When present, Meissner corpuscle capsules were CD34⁺ but often poorly defined (Fig. 5M). The capsules of deep dermal Krause and genital corpuscles displayed CD34 (Fig. 6B and D), Glut-1 (Fig. 6E to G), and α-SMA (Fig. 6J) immunoreactivity. Table 2 summarizes the immunohistochemical properties of the WT1⁺ sensory corpuscles documented in this study using our antibody panel.

Table 2.

Immunohistochemical Properties of WT1 ⁺ Sensory Corpuscles Identified in This Study.

Marker	Pacinian Corpuscles	Golgi-Mazzoni Corpuscles	Meissner Corpuscles	Ruffini-like Corpuscles	Genital and Krause Corpuscles
α-SMA	Capsule – –/+	–	–	–	Capsule (deep dermal type) ±
CD34	Intermediate layer +	Inner core + Irregular intermediate layer + Capsule –	Capsule ±	Capsule ±	Capsule (deep dermal type) +
Glut-1	Outer core +	Capsule +	–	N/A	Capsule (deep dermal type) ±
Nestin	Inner core Schwann cells +	Inner core Schwann cells +	Schwann cells +	Schwann cells +	Schwann cells +
NF	Axons +	Axons +	Axons +	Axons +	Axons +
NSE	Axons +	Axons +	Axons +	Axons +	Axons +
S100	Inner core Schwann cells +	Inner core Schwann cells +	Schwann cells +	Schwann cells +	Schwann cells +
SOX10	Inner core Schwann cells +	Inner core Schwann cells +	Schwann cells +	Schwann cells +	Schwann cells +
TH	N/A	N/A	N/A	N/A	N/A
WT1	Inner core Schwann cells +	Inner core Schwann cells +	Schwann cells +	Schwann cells +	Schwann cells +

This table presents the specific corpuscular components that exhibited immunoreactivity, along with the approximate percentages of corpuscles displaying these immunoreactivities. The immunohistochemical characteristics of penile sensory corpuscles remained consistent across different anatomical locations, including the prepuce, glans, and penile bulb.

+

Marker positive in most corpuscles (>90%).

±

Marker positive in a moderate subpopulation of corpuscles (~ 40–60%).

– –/+

Marker positive in a small subpopulation of corpuscles (~10–20%).

–

Marker not detected in all corpuscles.

N/A Antibody not applied to sensory corpuscle.

WT1 Immunohistochemical Staining in FNEs and Nerve Bundles

FNEs were highly dense in both the prepuce and glans, with frequent detection of intraepithelial nerve fibers (not shown). With signal amplification, nmSCs of preputial FNEs exhibited weak to strong (typically weak) WT1 immunoreactivity associated with the NF⁺ axons (Fig. 7A to F). Preputial nerve bundles were densely distributed throughout the prepuce and displayed a characteristic structure, with a Glut-1⁺ compact perineurium, CD34⁺ endoneurial cells, SOX10⁺, S100⁺, and nestin⁺ Schwann cells, and NF⁺, NSE⁺, and TH⁺ axons. Preputial nerve bundles exhibited two distinct WT1 immunolocalizations, consistently observed exclusively in mSCs within the same nerve bundles (Fig. 8A to L). Signal amplification was often required to enhance immunostaining from weak or moderate to strong. In most cases, WT1 expression was localized to the Schwann cell cytoplasm surrounding the axons, characterized by Cajal band-like staining in the internodal regions (arrows in Figs. 3F and 8A, C, F, I, J, and L), perinuclear staining (arrowheads in Fig. 8A, G, H, J, and K), and Schmidt–Lanterman incisure staining also within the internodal regions (arrows in Fig. 8G). In other instances, within the same nerve bundles, WT1 displayed myelin-associated staining, particularly evident in transversely sectioned axons. Unmyelinated noradrenergic axons in nerve bundles exhibited selective TH immunoreactivity (Fig. 8C and I), and double immunohistochemistry with WT1 confirmed the absence of WT1 expression in their associated nmSCs. Both mSCs and nmSCs within nerve bundles exhibited selective SOX10 nuclear immunostaining (Fig. 8G, J, and K).

Figure 7.

Preputial free nerve endings (FNEs) with WT1 expression in non-myelinating Schwann cells, as shown by double immunohistochemistry. (A–F) FNEs in the papillary dermis, with NF⁺ axons (brown) associated with WT1⁺ Schwann cell cytoplasm (magenta). The staining intensity is weak to strong. Scale bars = 50 µm (A–F).

Figure 8.

Preputial nerve bundles with WT1 expression in Schwann cells, demonstrated through single and double immunohistochemistry. (A, B) Serially sectioned nerve bundles with axons in cross-section: (A) WT1 exhibits a punctate or dot-like expression pattern characteristic of Cajal bands along the outermost (abaxonal) Schwann cell compartment of numerous myelinated axons (arrows). In some axons, WT1 is restricted to the perinuclear Schwann cell cytoplasm (arrowhead), whereas in others, staining appears circumferential around the internodal myelin sheath; (B) CD34 highlights endoneurial fibroblasts and surrounding dermal fibroblasts (brown). Myelinated and unmyelinated axons within the nerve bundles both display strong NF immunostaining (magenta). (C) Nerve bundle with WT1⁺ Schwann cells exhibiting punctate periaxonal Cajal band-like expression (brown, arrows). TH⁺ unmyelinated axons (magenta) are present but unassociated with WT1⁺ Schwann cells. (D, E) Serially sectioned S-shaped nerve bundle: (D) WT1 immunostaining highlights the longitudinal bands of Schwann cell cytoplasm surrounding longitudinally sectioned axons; (E) CD34 labels endoneurial fibroblasts and surrounding dermal dendrocytes (brown). NF⁺ myelinated and unmyelinated axons are visible within the bundle (magenta). (F) Consecutive serial section of the same nerve bundle shown in C, with axons in cross-section showing strong NF immunostaining in both myelinated and unmyelinated axons (brown). WT1 expression (magenta) is restricted to Schwann cells of myelinated axons, with staining patterns ranging from circumferential internodal myelin to the characteristic punctate pattern associated with Cajal bands (arrow). (G) Nerve bundle predominantly containing longitudinally sectioned axons. WT1 immunopositivity is observed in Schwann cell cytoplasmic channels along the myelinated axons, including perinuclear staining (arrowhead) and Schmidt–Lanterman incisures (arrows). Schwann cell nuclei are SOX10⁺ (magenta). (H) Nerve bundle with longitudinally sectioned NF⁺ axons (brown). WT1 expression (magenta) is observed in the perinuclear (arrowhead) and internodal Schwann cell cytoplasm. (I) Two small nerve bundles with axons in cross-section. WT1 (brown) exhibits a characteristic punctate Cajal band-like staining pattern in the Schwann cell cytoplasm (arrow). TH immunoreactivity is selectively present in unmyelinated axons (magenta), which are unassociated with WT1⁺ Schwann cells. Two small blood vessels in the field also show WT1⁺ endothelial cells (brown) and TH⁺ autonomic innervation (magenta). (J, K) Nerve bundles displaying WT1⁺ Schwann cells (brown) with perinuclear (arrowheads) and internodal Cajal band-like staining (arrows), along with SOX10⁺ Schwann cell nuclei (magenta). (L) Nerve bundle exhibiting the typical dot-like WT1⁺ (brown) Schwann cell staining pattern associated with Cajal bands (arrows) and a well-defined Glut-1⁺ perineurium (magenta). Scale bars = 50 µm (A–C, F–L), 100 µm (D, E).

Double Immunofluorescence Staining for WT1/Nestin and WT1/S100

Confocal laser scanning analysis of double immunofluorescence staining revealed no colocalization of WT1 and S100 in the Schwann lamellar cells of sensory corpuscles (Fig. 9A to D and I to L). In contrast, WT1 and nestin colocalized within these lamellar cells (Fig. 9E to H and M to T). The same pattern was observed in mSCs within nerve bundles, where WT1 and S100 showed no colocalization (Fig. 10A to C), whereas WT1 and nestin colocalized (Fig. 10D to I). Occasionally, merged confocal images of transversely sectioned axons revealed a linear WT1⁺ and nestin⁺ staining pattern, resembling the appositions between Cajal bands (white arrowheads in Fig. 10F and I). Figure 11 presents a simplified diagrammatic summary of our study’s findings on WT1 expression in Schwann cells of nerve bundles, sensory corpuscles, and FNEs.

Figure 9.

Double immunofluorescence analysis of WT1, nestin, and S100 in Schwann cells of preputial genital, Pacinian, and Meissner corpuscles. (A–D) Genital corpuscle corresponding to the corpuscle in Fig. 6I, in a consecutive serial section: (A) WT1 staining in the lamellar cell cytoplasm (red); (B) S100 staining in the same cell compartment (green); (C) DAPI nuclear staining (blue); (D) although WT1 and S100 are coexpressed in these Schwann cells with a typical cytoplasmic staining pattern, the merged image shows no colocalization, as indicated by the absence of yellow. (E–H) The same genital corpuscle shown in A–D, in the next consecutive section: (E) WT1 staining in the lamellar cell cytoplasm (red); (F) nestin staining in the same cell compartment (green); (G) DAPI nuclear staining (blue); (H) merged image showing colocalization of WT1 and nestin in the Schwann cell cytoplasm (yellow). (I–L) Inner core of a Pacinian corpuscle corresponding to the Pacinian in Fig. 1L, in a consecutive serial section: (I) WT1 staining in the lamellar cell cytoplasm (red); (J) S100 staining in the same cell compartment (green); (K) DAPI nuclear staining (blue); (L) merged image showing no colocalization of WT1 and S100, as indicated by the absence of yellow. (M–P) Inner core of the same Pacinian corpuscle shown in I–L, in the next consecutive section: (M) WT1 staining in the lamellar cell cytoplasm (red); (N) nestin staining in the same cell compartment (green); (O) DAPI nuclear staining (blue); (P) merged image demonstrating colocalization of WT1 and nestin in the Schwann cell cytoplasm (yellow). (Q–T) Meissner corpuscle: (Q) WT1 expression in Schwann or lamellar cells (red); (R) nestin expression in the same cells (green); (S) DAPI nuclear staining (blue); (T) merged image showing colocalization of WT1 and nestin in the Schwann cell cytoplasm (yellow). Scale bars = 25 µm (A–T).

Figure 10.

Double immunofluorescence analysis of WT1, nestin, and S100 in Schwann cells of preputial nerve bundles. (A–C) Longitudinally sectioned nerve bundle: (A) WT1 staining of Schwann cell cytoplasmic channels (red); (B) S100 staining in the same compartment (green); (C) although WT1 and S100 are coexpressed in these Schwann cells, the merged image reveals no colocalization of WT1 and S100, as indicated by the absence of yellow. (D–F) Longitudinally sectioned nerve bundle: (D) WT1 staining of Schwann cell cytoplasmic channels (red); (E) nestin staining in the same cell compartment (green); (F) merged image demonstrating colocalization of WT1 and nestin (yellow). Cajal band-like staining is observed in transversely sectioned axons (arrow), along with a more linear pattern resembling the appositional regions flanking Cajal bands (arrowhead). In addition, a staining pattern similar to that of Schmidt–Lanterman incisures is present (green arrowhead). (G–I) Transversely sectioned nerve bundle: (G) WT1 expression in Schwann cell cytoplasmic channels (red); (H) nestin staining of the same cell compartment (green); (I) merged image showing colocalization of WT1 and nestin (yellow). Note the Cajal band-like staining in transversely sectioned axons (arrows), along with a linear staining pattern that more closely resembles the appositional regions flanking Cajal bands (arrowhead). Scale bars = 12 µm (A–C, G–I), 16 µm (D–F).

Figure 11.

Summary of WT1 immunohistochemical expression in Schwann cell subtypes identified in this study. Peripheral nerve bundles contained a variable mixture of myelinated and unmyelinated axons, associated with WT1⁺ myelinating Schwann cells (mSCs) and WT1^– non-myelinating Schwann cells (nmSCs), respectively. These nerve bundles gave rise to (i) sensory corpuscles containing modified WT1⁺ nmSCs; (ii) free nerve endings (FNEs) associated with WT1⁺ nmSCs. Abbreviations: mSCs, myelinating Schwann cells; nmSCs, non-myelinating Schwann cells; FNEs, free nerve endings.

Discussion

This study provides the first characterization in the scientific literature of WT1 immunohistological expression in the human mechanosensory corpuscles. Specifically, we demonstrated WT1 expression in corpuscular receptors of the adult human penile prepuce, glans, and bulb, displaying a typical Schwann cell cytoplasmic staining pattern. Pacinian, Golgi-Mazzoni, Meissner, genital, Krause, and Ruffini-like corpuscles in the human penis all exhibited WT1 expression, though at varying levels. WT1 exhibited moderate to strong expression in Pacinian, Golgi-Mazzoni, genital, Krause, and Ruffini-like corpuscles, which are located deep in the dermis and preputial dartos layer. WT1⁺ Pacinians were also identified deep within the spongiosal erectile tissue of the penile bulb. In contrast, Meissner corpuscles, situated superficially within dermal papillae, generally showed weak WT1 staining, often requiring signal amplification to achieve moderate or strong staining. This suggests that WT1 expression levels in penile sensory corpuscles are influenced by their histological position, with higher expression in deeper corpuscles and lower expression in those located more superficially. In addition, we documented WT1 expression in nmSCs associated with FNEs and in mSCs within nerve bundles. Figure 11 provides a schematic overview of our results on WT1 expression across Schwann cell types.

Apart from WT1, we utilized a diverse antibody panel to characterize the molecular profiles of WT1⁺ neural structures and assess potential colocalization patterns. This battery included markers for endoneurium (CD34), perineurium (α-SMA, Glut-1), Schwann cells (nestin, S100, SOX10), and axons (neurofilaments, NSE, TH). This immunohistochemical exploration confirmed that WT1⁺ sensory corpuscles and nerve bundles exhibit well-documented molecular profiles consistent with those described in the literature.^5,8,15 Table 2 summarizes the immunoreactivity patterns observed in sensory corpuscles in this study. Beyond WT1, several additional novel findings from our immunohistochemical characterization of sensory corpuscles warrant special mention and will be discussed at the end of the Discussion section to maintain focus on WT1.

Furthermore, double immunofluorescence and confocal microscopy revealed WT1 colocalization with nestin while excluding colocalization with S100 in Schwann cells of both sensory corpuscles and nerve bundles. This finding suggests distinct subcellular distributions or differential regulatory interactions among these markers, potentially reflecting functional specialization within Schwann cells. For example, WT1’s colocalization with nestin might suggest a possible crosstalk with the intermediate filament cytoskeleton, potentially influencing Schwann cell cytoskeletal dynamics. Notably, while some authors ⁶⁴ report that nestin is expressed in only a small subset of Schwann cells in Meissner and Pacinian corpuscles of human palmar digital skin, our findings indicate that both nestin and WT1 are present in Schwann cells of nearly all sensory corpuscles, at least in the penis. Crucially, Wagner et al. ⁶⁵ showed that WT1 is an activator of nestin expression, with both molecules coexpressed in the same cells in developing and adult podocytes, the epicardium, and developing coronary vessels. Vasuri et al. ⁶⁶ later demonstrated cytoplasmic colocalization of WT1 and nestin in endothelial cells of small-sized vasa vasorum in normal human arteries, supporting WT1’s role as a post-transcriptional activator of nestin. Another study by this group also demonstrated WT1 and nestin immunopositivity in endothelial cells of neovessels within atheromatous plaques. ⁶⁷ Scholz et al. ⁶⁸ postulated that WT1 contributes to cytoskeletal remodeling in vascular cells by activating nestin gene transcription as part of the response to myocardial ischemia. Together, this evidence supports the idea that WT1 could regulate nestin expression in Schwann cells and might indirectly influence Schwann cell cytoskeletal dynamics. Assessing the potential colocalization of WT1 with vimentin in Schwann cells could further substantiate this hypothesis.

The subcellular localization of WT1 has been mapped to actively translating polysomes in the cytoplasm of various WT1-expressing cell lines, ³⁸ while certain WT1 isoforms predominantly localize to nuclear speckles.^69,70 As noted in our Introduction, the dual nuclear and cytoplasmic localization of WT1 has been attributed to its nucleo-cytoplasmic shuttling properties. ³⁸ The immunohistological detection of these localizations depends on the use of specific antibodies, targeting either the N-terminal or C-terminal regions of the WT1 protein. To elucidate the exact subcellular localization(s) of WT1 across Schwann cell subtypes identified in this study, advanced ultrastructural approaches, such as immunogold electron microscopy, are warranted.

We also confirmed the WT1 immunohistochemical expression in mSCs of peripheral nerve bundles, as previously described by Schittenhelm et al. ⁴⁰ and Inagaki et al., ⁴¹ exhibiting typical perinuclear, Cajal band and myelin staining patterns. However, we also identified WT1 expression in Schmidt–Lanterman incisures, a feature not previously documented. While Schittenhelm et al. ⁴⁰ and Inagaki et al. ⁴¹ both presented microphotographs of WT1⁺ nerve bundles, they did not identify or explicitly discuss the Cajal band–like staining pattern, which we have now incorporated into our analysis. Established molecular markers for Schmidt–Lanterman incisures include E-cadherin^47,71 and CNPase, ⁷² whereas markers of Cajal bands include merlin, ⁷³ vimentin, ⁴⁴ plectin, ⁴⁴ and utrophin, ⁷⁴ among others. To our knowledge, only one study has reported nestin immunolocalization in Cajal bands and the absence of colocalization between nestin and S100 in these structures, using teased rat sciatic nerves. ⁷⁵ Given that we demonstrated WT1 and nestin colocalization in mSCs, along with the lack of WT1 colocalization with S100 in mSCs, these findings are consistent with and further support our confocal colocalization analyses.

Our results suggest that WT1 may serve as a marker for both Cajal bands and Schmidt–Lanterman incisures, though signal amplification may be necessary for optimal immunostaining of these complex Schwann cell cytoplasmic motifs. As we occasionally observed a more linear staining pattern in transversely sectioned axons in merged confocal images, resembling the appositions between Cajal bands (see Fig. 1E in Sherman et al.) ⁴⁷ , further analysis is needed to determine whether WT1 and nestin specifically localize to Cajal bands or are also present in adjacent appositional regions, where the cytoplasm is compressed between the abaxonal Schwann cell myelin and the overlying plasma membrane. More broadly, refined analyses using teased fiber preparations are needed to precisely define the morphological and molecular subdomains within the axoglial apparatus where WT1 immunolocalizes (node of Ranvier, paranode, juxtaparanode, internode, and autotypic tight junctions of mSCs).^76,77

As a rule, WT1 was absent in nmSCs within nerve bundles, as confirmed through double immunohistochemistry for WT1 and TH, a specific marker of unmyelinated autonomic sympathetic axons. These sections consistently showed TH⁺ axons without association with WT1⁺ cells. In contrast, double immunohistochemistry for WT1 and NF (a general marker of both myelinated and unmyelinated axons) revealed that larger-diameter axons were specifically associated with WT1⁺ mSCs. Similarly, double immunohistochemistry for WT1 and SOX10 (a general nuclear marker of Schwann cells) revealed that SOX10⁺ Schwann cell nuclei were exclusively associated with WT1⁺ mSCs. Thus, WT1’s absence in nmSCs within nerve bundles suggests it is involved in the myelination machinery and that WT1 expression in peripheral nerves might be regulated by signaling cues specific to myelinated axons.

Moreover, Inagaki et al. ⁴¹ reported weak WT1 staining in the Schwann cell cytoplasm and myelin of cauda equina nerve roots but did not explore the significance of this dual WT1 expression pattern (cytoplasmic and myelin-associated). This dual distribution likely reflects functional heterogeneity among mSCs and suggests distinct roles for WT1 in their biology. For example, WT1 might be involved in pathways regulating peripheral myelin production, maintenance, or remodeling. Colocalization studies with myelin markers (e.g., MBP, MPZ/P0, PLP1, PMP22, MAG) could help determine whether WT1 expression is associated with specific stages of myelin production, maintenance, or remodeling. At the same time, the WT1 cytoplasmic location suggests functions beyond myelin dynamics, involving Schwann cell differentiation, maintenance, cytoskeletal organization, plasticity, or axon–glia interactions. Given WT1’s known functions in cell proliferation, migration, and plasticity, ⁷⁸ its presence in the Schwann cell cytoplasm could indicate involvement in peripheral nerve regeneration or response to injury, possibly contributing to Büngner band formation.

Another novel finding of this study is that Schwann cells associated with preputial FNEs also express low levels of WT1, as indicated by their weak to strong staining (typically weak), which required signal amplification for detection. This WT1⁺ Schwann cell population associated with preputial FNEs corresponds to nmSCs. In our previous study, ¹⁵ we also demonstrated that Schwann cells associated with preputial FNEs express SOX10, a marker of Schwann cell lineage and stem-like properties.^{79 –81} Notably, a subset of dermal FNEs is thought to originate not directly from the neural crest but from boundary cap cells, a transient population of multipotent cells derived from the neural crest, which aggregates within the spinal nerve roots. ¹³ In addition, a population of boundary cap–derived dermal FNEs retains multipotency, functioning as a stem cell–like population. ¹³ WT1 has been shown to play an intrinsic role in mesenchymal stem cell lineages. ²⁵ Based on this evidence, it is tempting to speculate that SOX10⁺ and WT1⁺ nmSCs associated with adult human preputial FNEs may exhibit stem cell–like properties. Of note, cutaneous SOX10⁺ Schwann cells associated with dermo-epidermal FNEs have also been implicated in mechanotransduction, specifically in nociception. ⁸²

In all neural structures examined in this study (nerve bundles, sensory corpuscles, and FNEs), axons displayed no WT1 immunoreactivity. A relevant immunohistochemical study by Coosemans et al. ⁸³ reported WT1 upregulation in neuronal tissue identified within paraffin-embedded sections of deep endometriotic lesions, suggesting this may result from a defect in the NGF-TrkA-Src/Ras pathway. They also observed negative or very weak WT1 immunopositivity in isolated normal peripheral nerves, which contrasts with our findings of WT1 expression in mSCs of nerve bundles. This discrepancy may be attributed to their lack of signal amplification and the use of archival paraffin-embedded tissues, which could have affected WT1 detectability. Although Parenti et al. ⁴² reported WT1⁺ Schwann cells in human fetal peripheral nerves using light microscopic immunohistochemistry, determining whether WT1 immunoreactivity is strictly confined to Schwann cells or also extends to axons can be challenging in fetal specimens based solely on immunohistochemistry. Therefore, studies using double immunofluorescence with WT1 and axonal-specific markers would be valuable in clarifying whether WT1 is transiently expressed in peripheral axons during certain stages of fetal development before undergoing rapid downregulation. Future studies should also investigate the fetal age at which WT1 first becomes immunohistochemically detectable in immature sensory corpuscles. Preliminary unpublished data from us suggest that WT1 expression in the inner core of fetal penile Pacinian corpuscles begins at approximately 17–20 weeks of gestation, whereas other sensory corpuscle types develop much later in fetal life and/or postnatally. ⁸⁴

Together, these results suggest that WT1 is expressed at varying immunohistochemical levels in Schwann cells, extending from those in the proximal spinal nerve roots (as reported by Inagaki et al. ⁴¹ in cauda equina nerve roots) through more distal peripheral nerves ⁴⁰ to those in sensory corpuscles and FNEs, as demonstrated in our current study (Fig. 11). The presence of WT1 in Schwann cells of peripheral nerves, sensory corpuscles, and FNEs suggests a functional role that may relate to their development, maintenance, or specialized interactions with axons. The potential physiologic implications are far-reaching, but functional studies are needed to determine the specific roles WT1 may play in these processes. Overall, these results open new avenues for exploring WT1’s molecular mechanisms and potential roles in peripheral glia throughout both developmental and mature stages, extending beyond its well-established functions in non-neural tissues.^{25,26,85 –89}

Our novel finding of WT1 expression in corpuscular Schwann cells was not unexpected, as sensory corpuscles are essentially the distal-most extensions of peripheral nerve components, and WT1⁺ mSCs in peripheral nerves had already been reported by Schittenhelm et al. ⁴⁰ and Inagaki et al. ⁴¹ It is important to note that the weak WT1 immunostaining of Schwann cells reported by these authors may be attributed to the absence of signal amplification methods in their studies. In contrast, our use of optimized signal amplification with the EnVision FLEX Mouse Linker reagent (Dako-Agilent) resulted in strong, crisp WT1 immunostaining with an excellent signal-to-noise ratio, as demonstrated in our microphotographs. These signal amplification techniques, which incorporate secondary linker antibodies, are a standard practice in immunohistochemistry with Dako-Agilent ⁹⁰ and other ⁹¹ automated platforms, widely used in both basic research and tumor pathology. Importantly, in numerous cases where signal amplification was applied for WT1 and other markers, we compared the immunostains with serial or nearby sections without signal amplification to assess the signal-to-noise ratio. We encountered no negative issues using these techniques, though careful interpretation remains essential when assessing the expression levels of the target antigen. In the case of WT1 in mSCs of nerve bundles, immunohistochemical expression was weak to moderate (typically moderate) without signal amplification and typically strong with signal amplification.

Our results should be interpreted in the context of a series of studies published over the past 25 years, which have documented important functions of WT1 specifically in the nervous system.^{28 –35} WT1 is essential for retinal development, as its loss leads to retinal ganglion cell apoptosis, impaired optic nerve growth, and disrupted expression of Pou4f2, a key transcription factor for ganglion cell differentiation. ²⁸ WT1 is also implicated in neuronal degeneration in Alzheimer’s disease, as it selectively immunostains a subset of neurofibrillary tangle-containing neurons, and its expression correlates with increased neuronal apoptosis in vitro. ³⁵ In the olfactory system, the WT1 (+KTS) isoform is required for proper epithelial development, regulating neurogenic factors such as Mash1 and neurogenin 1. ²⁹ WT1 also plays a crucial role in the development of the posterior taste field by controlling the expression of BMP4, Ptch1, and Lef1, which regulate circumvallate papillae formation and innervation. ³⁰ In the spinal cord, neuron-specific WT1 deletion disrupts locomotion and alters interneuron composition, affecting motor coordination. ³¹ WT1⁺ interneurons in the spinal cord further regulate left–right alternation during locomotion, as their inactivation leads to impaired contralateral limb coordination. ³² WT1 has also been identified as a key regulator of synaptic plasticity and memory flexibility in the hippocampus, acting as a transcriptional repressor that modulates memory retention and behavioral adaptability in mice. ³³ Last, WT1⁺ dB4 neurons in the caudoventral medulla oblongata are crucial for respiration, as their selective ablation results in neonatal lethality due to respiratory failure, highlighting its indispensable role in central respiratory control. ³⁴ Collectively, these findings establish WT1 as a critical regulatory molecule in neural development, function, and disease, with roles extending to sensory systems such as retinal ganglion cells, ²⁸ the olfactory epithelium, ²⁹ and the posterior taste field. ³⁰

These proposed multifaceted roles of WT1 in central neurons and sensory systems provide a broader framework for interpreting our findings in Schwann cells of penile sensory corpuscles, nerve bundles, and FNEs. Our study suggests previously unrecognized functions for WT1 in peripheral glial cells, where it may indirectly contribute to mechanosensation, axon–glia communication, and Schwann cell development, maintenance, and/or plasticity. The presence of WT1 in both mSCs and nmSCs, but not in axons, indicates glia-specific functions. Given WT1’s involvement in sensory processing across the visual, ²⁸ olfactory, ²⁹ and gustatory sensory systems, ³⁰ its expression in corpuscular Schwann cells suggests a broader regulatory function in peripheral mechanosensory pathways that warrants further investigation.

Beyond WT1, three additional findings from our immunohistochemical characterization of sensory corpuscles warrant special mention. First, α-SMA immunoreactivity in the capsules of Pacinian corpuscles was previously reported only by Cepeda-Emiliani et al. ¹⁵ In this study, we confirm that a subpopulation of Pacinian corpuscles exhibits capsular α-SMA immunopositivity. While its significance remains unclear, this finding suggests potential contractile properties of Pacinian capsular cells, similar to what has been proposed for perineurial cells based on the presence of α-SMA in the perineurium.^63,92

Second, we identified a subpopulation of deep dermal Krause and large genital corpuscles with Glut-1⁺, α-SMA⁺, and CD34⁺ capsules, suggesting a molecular continuity with the Glut-1⁺ peripheral nerve perineurium and the CD34⁺ peripheral nerve endoneurium. Among sensory corpuscles, Pacinian corpuscles are uniquely distinguished by their molecular continuity with both the CD34⁺ endoneurium, which forms the CD34⁺ intermediate layer, and the Glut-1⁺ perineurium of peripheral nerves, which forms the Glut-1⁺ outer core. ⁹³ Glut-1 is a well-established marker of perineurium, ⁶² while α-SMA, though not traditionally classified as a perineurial marker, is expressed in perineurial cells, with more intense labeling in the outer perineurial layers. ⁶³ However, recent immunohistochemical studies have not confirmed this perineurial molecular continuity in non-Pacinian corpuscles, such as Meissner and genital corpuscles.^16,94 In this study, we demonstrate that, similar to Pacinian corpuscles, a subpopulation of deep dermal penile sensory corpuscles contains CD34⁺ endoneurial-derived capsular cells and Glut-1⁺ and α-SMA⁺ perineurially derived capsular cells. This finding is currently being investigated in greater depth in a parallel study by our group.

Third, to the best of our knowledge, our study also provides the first immunohistochemical demonstration and characterization of Golgi-Mazzoni corpuscles in the human penile prepuce. The preputial Golgi-Mazzoni corpuscles we identified were nearly identical in structure to the Golgi-Mazzoni illustrated in Fig. 5.6 of Csillag’s atlas, ¹⁴ where he also notes that these corpuscles are typically found in the external genitalia, conjunctiva, and nail bed, as well as in the peritoneum, tendons, and joint capsule attachments, suggesting a likely proprioceptive function. Their molecular profile closely resembled that of Pacinian corpuscles, with the key difference that the CD34⁺ intermediate layer was often irregular, and the cells extended prolongations into the inner core. Whether these CD34⁺ cells, characterized by their thin and elongated cytoplasmic processes, are telocytes (as proposed by Díaz-Flores et al. ⁹⁵ for the CD34⁺ intermediate layer cells of Pacinian corpuscles and the CD34⁺ capsular cells of Meissner corpuscles) remains to be determined. Thus, similar to Pacinian corpuscles and the aforementioned subpopulation of deep dermal Krause and genital corpuscles, the Golgi-Mazzoni corpuscles also exhibit CD34⁺ endoneurial and Glut-1⁺ perineurial continuity with the nerve bundles supplying them. Although their densities were low, these corpuscles should not be considered rare or exotic but rather part of the normal spectrum of specialized corpuscular receptors in preputial tissues. ⁵⁹ After thoroughly analyzing a sufficient number of sections from each specimen, Golgi-Mazzoni corpuscles could be consistently identified in most specimens.

This study has several limitations, including limited functional insights, lack of developmental timepoints, restricted coexpression and colocalization analyses, potential antibody sensitivity issues, and specimens limited to penile tissues. First, while this study provides a detailed immunohistochemical characterization of WT1 in Schwann cells, it does not establish its functional role in sensory corpuscles or peripheral nerves. Future functional research should investigate whether WT1 contributes to Schwann cell differentiation, maintenance, or plasticity. Second, our findings are limited to adult tissues, and we did not assess WT1 expression across fetal or early postnatal developmental stages. Future studies should define WT1 expression timelines in different sensory corpuscle types using fetal specimens. Third, although we performed double immunohistology for WT1 with Schwann cell markers (S100, nestin, and SOX10), additional double immunofluorescence analyses with markers of Schwann cells, Cajal bands, and intermediate filaments would help clarify WT1’s role in these structures. Fourth, optimal WT1 detection required signal amplification in some cases, particularly in Meissner corpuscles and FNEs, suggesting that baseline WT1 expression may be low in certain corpuscle types or nerve fibers. Antibody sensitivity or tissue processing differences may have influenced the observed expression patterns, and further immunohistochemical studies should validate these findings. Finally, this study was conducted exclusively on human penile tissues. Although some preputial specimens showed signs of inflammation, the mechanosensory corpuscular receptors studied were not affected and maintained normal structures and molecular profiles. Comparative studies are needed to determine whether WT1 expression is site-specific or part of a broader neuroanatomical pattern (e.g., sensory corpuscles of palmar digital skin, extra-genital mucocutaneous regions, and internal organs).

In conclusion, this study provides the first immunohistological description of WT1 in the modified nmSCs of human mechanosensory corpuscles. As discussed, the potential implications of this finding are broad, particularly in understanding Schwann cell development, maintenance, or plasticity. However, further research is needed to elucidate the functional significance of WT1 in Schwann cell biology under both normal and pathological conditions of the peripheral nervous system.