A RET-ER81-NRG1 Signaling Pathway Drives the Development of Pacinian Corpuscles.

Axon-Schwann cell interactions are crucial for the development, function, and repair of the peripheral nervous system, but mechanisms underlying communication between axons and nonmyelinating Schwann cells are unclear. Here, we show that ER81 is functionally required in a subset of mouse RET+ mechanosensory neurons for formation of Pacinian corpuscles, which are composed of a single myelinated axon and multiple layers of nonmyelinating Schwann cells, and Ret is required for the maintenance of Er81 expression. Interestingly, Er81 mutants have normal myelination but exhibit deficient interactions between axons and corpuscle-forming nonmyelinating Schwann cells. Finally, ablating Neuregulin-1 (Nrg1) in mechanosensory neurons results in no Pacinian corpuscles, and an Nrg1 isoform not required for communication with myelinating Schwann cells is specifically decreased in Er81-null somatosensory neurons. Collectively, our results suggest that a RET-ER81-NRG1 signaling pathway promotes axon communication with nonmyelinating Schwann cells, and that neurons use distinct mechanisms to interact with different types of Schwann cells. SIGNIFICANCE STATEMENT: Communication between neurons and Schwann cells is critical for development, normal function, and regeneration of the peripheral nervous system. Despite many studies about axonal communication with myelinating Schwann cells, mostly via a specific isoform of Neuregulin1, the molecular nature of axonal communication with nonmyelinating Schwann cells is poorly understood. Here, we described a RET-ER81-Neuregulin1 signaling pathway in neurons innervating Pacinian corpuscle somatosensory end organs, which is essential for communication between the innervating axon and the end organ nonmyelinating Schwann cells. We also showed that this signaling pathway uses isoforms of Neuregulin1 that are not involved in myelination, providing evidence that neurons use different isoforms of Neuregulin1 to interact with different types of Schwann cells.

c-Maf is required for the development of dorsal horn laminae III/IV neurons and mechanoreceptive DRG axon projections.

Establishment of proper connectivity between peripheral sensory neurons and their central targets is required for an animal to sense and respond to various external stimuli. Dorsal root ganglion (DRG) neurons convey sensory signals of different modalities via their axon projections to distinct laminae in the dorsal horn of the spinal cord. In this study, we found that c-Maf was expressed predominantly in the interneurons of laminae III/IV, which primarily receive inputs from mechanoreceptive DRG neurons. In the DRG, c-Maf⁺ neurons also coexpressed neurofilament-200, a marker for the medium- and large-diameter myelinated afferents that transmit non-noxious information. Furthermore, mouse embryos deficient in c-Maf displayed abnormal development of dorsal horn laminae III/IV neurons, as revealed by the marked reduction in the expression of several marker genes for these neurons, including those for transcription factors MafA and Rora, GABA(A) receptor subunit α5, and neuropeptide cholecystokinin. In addition, among the four major subpopulations of DRG neurons marked by expression of TrkA, TrkB, TrkC, and MafA/GFRα2/Ret, c-Maf was required selectively for the proper differentiation of MafA⁺/Ret⁺/GFRα2⁺ low-threshold mechanoreceptors (LTMs). Last, we found that the central and peripheral projections of mechanoreceptive DRG neurons were compromised in c-Maf deletion mice. Together, our results indicate that c-Maf is required for the proper development of MafA⁺/Ret⁺/GFRα2⁺ LTMs in the DRG, their afferent projections in the dorsal horn and Pacinian corpuscles, as well as neurons in laminae III/IV of the spinal cord.

The development of human digital Meissner's and Pacinian corpuscles.

Meissner's and Pacinian corpuscles are cutaneous mechanoreceptors responsible for different modalities of touch. The development of these sensory formations in humans is poorly known, especially regarding the acquisition of the typical immunohistochemical profile related to their full functional maturity. Here we used a panel of antibodies (to specifically label the main corpuscular components: axon, Schwann-related cells and endoneurial-perineurial-related cells) to investigate the development of digital Meissner's and Pacinian corpuscles in a representative sample covering from 11 weeks of estimated gestational age (wega) to adulthood. Development of Pacinian corpuscles starts at 13 wega, and it is completed at 4 months of life, although their basic structure and immunohistochemical characteristics are reached at 36 wega. During development, around the axon, a complex network of S100 positive Schwann-related processes is progressively compacted to form the inner core, while the surrounding mesenchyme is organized and forms the outer core and the capsule. Meissner's corpuscles start to develop at 22 wega and complete their typical morphology and immunohistochemical profile at 8 months of life. In developing Meissner's corpuscles, the axons establish complex relationships with the epidermis and are progressively covered by Schwann-like cells until they complete the mature arrangement late in postnatal life. The present results demonstrate an asynchronous development of the Meissner's and Pacini's corpuscles and show that there is not a total correlation between morphological and immunohistochemical maturation. The correlation of the present results with touch-induced cortical activity in developing humans is discussed.

Abnormal development of pacinian corpuscles in double trkB;trkC knockout mice.

Pacinian corpuscles depend on either Aalpha or Abeta nerve fibers of the large- and intermediate-sized sensory neurons for the development and maintenance of the structural integrity. These neurons express TrkB and TrkC, two members of the family of signal transducing neurotrophin receptors, and mice lacking TrkB and TrkC lost specific neurons and the sensory corpuscles connected to them. The impact of single or double targeted mutations in trkB and trkC genes in the development of Pacinian corpuscles was investigated in 25-day-old mice using immunohistochemistry and ultrastructural techniques. Single mutations on trkB or trkC genes were without effect on the structure and S100 protein expression, and caused a slight reduction in the number of corpuscles. In mice carrying a double mutation on trkB;trkC genes most of the corpuscles were normal with a reduction of 17% in trkB-/-;trkC+/- mice, and 8% in trkB +/-;trkC -/- mice. Furthermore, a subset of the remaining Pacinian corpuscles (23% in trkB-/-;trkC+/- mice; 3% in trkB+/-;trkC-/- mice) were hypoplasic or atrophic. Present results strongly suggest that the development of a subset of murine Pacinian corpuscles is regulated by the Trk-neurotrophin system, especially TrkB, acting both at neuronal and/or peripheral level. The precise function of each member of this complex in the corpuscular morphogenesis remains to be elucidated, though.

Ontogeny of the cutaneous sensory organs.

The ontogeny of cutaneous sensory nerve organs is described in higher vertebrates, and includes the lamellated corpuscles of Meissner, Pacini and Herbst, and the Merkel cell-neurite complex with bird Merkel and Grandry corpuscles, and mammalian Merkel cells. The main common feature is that for most corpuscles there is an inside-out order of assembly around the nerve ending which is present from the beginning of end-organ ontogeny. The exception is the mammalian Merkel cell which is present in the epidermis before the entrance of nerve fibers, and could play a promotional role in the development of skin innervation. The developmental origin of Herbst and Merkel corpuscles in birds is reported as demonstrated using embryological experiments with cell markers. Conclusions are that inner bulb cells of Herbst corpuscles and bird Merkel cells are of neural crest origin, whereas other cells (inner space and capsular cells for Herbst corpuscle and capsular cells for Merkel corpuscles) are provided by the local mesenchyme. The question of the ontogeny of mammalian Merkel cells is discussed in relation to the two debated hypothesis of epidermal and neural crest origins. Morphogenetic interactions during the development of cutaneous sensory end organs are also discussed.

Postnatal developmental changes in the expression of ErbB receptors in murine Pacinian cospucles.

Neuregulins and their signaling ErbB receptors play a critical role during the development of the mammalian peripheral nervous system, including some kinds of mechanoreceptors such as the Pacinian corpuscles which become structurally and functionally mature postnatally. In this study, we investigated whether or not ErbBs in Pacinian corpuscles undergoes developmental changes, as well as if their expression depends on the innervation. Pacinian corpuscles from 7-day- and 3-month-old mice were assessed for the immunohistochemical detection of EGFR or ErbB1, ErbB2, ErbB3 and ErbB4. The effect of denervation on the expression of ErbBs in mature Pacinian corpuscles was also analyzed. Developing 7-day-old Pacinian corpuscles express ErbB2 and ErbB3 immunoreactivity in the inner-core (regarded as modified Schwann cells), whereas the mature 3-month-old Pacinian corpuscles exclusively displayed ErbB4 immunoreactivity in the outer core and the capsule (regarded as endoneurial and perineurial cells). Denervation was without effect on the ErbB expression. Present results demonstrate maturational related changes and cell segregation in the expression of ErbB receptors by murine Pacinian corpuscles, and that this expression is independent of the innervation.

ETS transcription factor ER81 is required for the Pacinian corpuscle development.

ER81, a member of the ETS family of transcription factors, is involved in processes of specification of neuronal identity, control of sensory-motor connectivity, and differentiation of muscle spindles. Spindles either degenerate or are abnormal in mutant mice lacking ER81. We examined whether ER81 is required for the development of another class of mechanoreceptors, the Pacinian corpuscle. ER81 was expressed by the inner core cells of the corpuscles, as reflected by expression of the lacZ reporter gene in Er81(+/lacZ) mutants, thereby suggesting a role for ER81 in the corpuscle development. No Pacinian corpuscles or their afferent nerve fibers were present in the crus of Er81 null mice at birth. Legs of mutant embryos examined at E16.5 were also devoid of the corpuscles, but not of their afferents. Thus, Pacinian corpuscles do not form, and their afferents do not survive, in the absence of ER81. A deficiency of dorsal root ganglia neurons expressing calretinin, a marker for neurons subserving Pacinian corpuscles, correlated with the absence of corpuscles and their afferents in Er81 null mice. These observations indicate a requirement for ER81 in the assembly of Pacinian corpuscles and the survival of the sensory neurons that innervate them.

Reinnervation of rat Pacinian corpuscles after nerve crush during the postcritical period of development.

The ultrastructure of crural Pacinian corpuscles was examined after sciatic nerve crush performed in 7- to 20-day-old rats, i.e. during the postcritical period of development when the corpuscles no longer degenerate after axotomy but cease growing. The aim of our study was to assess the innervation pattern and structural changes of the corpuscles following transient denervation and subsequent reinnervation during their maturation and growth. Reinnervated corpuscles were examined by electron microscopy from 2.5 months after nerve crush onwards. After sciatic nerve crush at 7 days of age, the corpuscles are mostly reinnervated with multiple axon terminals, each of them enclosed within a newly formed inner core. The axial multiple cores are in part covered by a layer of concentric inner core lamellae and surrounded by a capsule, both structures having survived from the original corpuscle. After nerve crush at 10 days of age, reinnervated Pacinian corpuscles usually contain, in their axial region, a denervated remainder of the original core together with a few regenerated axon terminals enclosed within new inner cores. These axial structures are surrounded by a layer of concentric lamellae of the original core which may accommodate some regenerated terminals. Additional axon terminals with their small inner cores may be found at the outer aspect of the composite core beneath the capsule. When the nerve is crushed in 15-day-old rats, the inner core which is already well developed remains preserved by the time of reinnervation, and regenerating axons grow in between the original lamellae inducing only moderate neoformation of 2-3 lamellar layers which enclose the terminals. After crushing the sciatic nerve in 20-day-old rats, formation of new inner core lamellae is minimal and regenerated terminals become accommodated between the original lamellar of the core as is the case in adult animals. Regeneration of new inner cores and reinnervation of the preserved lamellar structure thus characterize the recovery of Pacinian corpuscles following reinnervation after nerve crush during the postcritical period of their development.

Pacinian corpuscle development involves multiple Trk signaling pathways.

The development of crural Pacinian corpuscles was explored in neonatal mutant mice lacking nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) or neurotrophin-4 (NT4), or their cognate Trk receptors. Deficits of the corpuscles and their afferents were greatest in NT3, less in BDNF, and least in NT4 null mice. Deletion of NGF or p75(NTR) genes had little or no impact. No Pacinian corpuscles were present in NT3;BDNF and NT3;NT4 double or NT3;BDNF;NT4 triple null mice. Deficits were larger in NT3 than TrkC mutants and were comparable to deficits observed in TrkB or TrkA mutants. Afferents of all corpuscles coexpressed TrkA and TrkB receptors, and some afferents coexpressed all three Trk receptors. Our results suggest that multiple neurotrophins, in particular NT3, regulate the density of crural Pacinian corpuscles, most likely by regulating the survival of sensory neurons. In addition, NT3/TrkB and/or NT3/TrkA signaling plays a greater role than NT3/TrkC signaling in afferents to developing Pacinian corpuscles.

Development of Meissner-like and Pacinian sensory corpuscles in the mouse demonstrated with specific markers for corpuscular constituents.

The development of Meissner-like and Pacinian corpuscles was studied in mice [from postnatal day (Pd) 0 to 42] by using immunohistochemistry for specific corpuscular constituents. The battery of antigens investigated included PGP 9.5 protein and neurofilaments, as markers for the central axon; S100 protein, vimentin, and p75(LNGFR) protein, to show Schwann-related cells; and epithelial membrane antigen to identify perineurial-related cells. In Meissner-like corpuscles immunoreactivity (IR) for neuronal markers was found by Pd7 and later. The lamellar cells of these corpuscles expressed first S100 protein IR (Pd7 to Pd42), then vimentin IR (Pd12 to Pd42), and transitory p75(LNGFR) IR (Pd7 to Pd19-20). Vimentin IR, but not epithelial membrane antigen, was detected in the capsule-like cells of the Meissner-like corpuscles. On the other hand, the density of Meissner-like corpuscles progressively increased from Pd0 to Pd19-20. Pacinian corpuscles were identified by Pd7. From this time to Pd42 the central axon showed IR for neuronal markers, and the inner core cells were immunoreactive for S100 protein. Moreover, vimentin IR was detected in the inner core cells by Pd19 and later. Unexpectedly, the central axons displayed S100 protein IR (from Pd7 to P28), while p75(LNGFR) protein IR or epithelial membrane antigen IR were never detected. Taken together, and based on the expression of the assessed antigens alone, the present results suggest that the Meissner-like and the Pacinian corpuscles in mice become mature around Pd19-Pd28 and Pd20, respectively. Furthermore, these results provide a baseline timetable for future studies in the normal or altered development of sensory corpuscles in mice since specific sensory corpuscles are functionally associated with different subtypes of sensory neurons the development of which is selectively disturbed in genetically manipulated mice.