Sequential Observation of Morphogenesis of Cutaneous Nerve Fibers in Fetal Rat Skin: An Immunohistochemical and Electron Microscopic Study Tadanori Inoue,

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Sequential Observation of Morphogenesis of Cutaneous Nerve Fibers in Fetal Rat Skin: An Immunohistochemical and Electron Microscopic Study Tadanori Inoue, Motoyuki Mihara Journal of Investigative Dermatology Volume 123, Issue 4, Pages 763-770 (October 2004) DOI: 10.1111/j.0022-202X.2004.23434.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Thirteenth fetal day Histology and immunohistochemistry. (A) Periderm cells and basal cells of the epidermis can be identified easily, and both the cells are flattened in shape. The basal cells are arranged irregularly. The dermis is highly edematous where a number of undifferentiated mesenchymal cells are diffusely distributed, whose nuclei were spindle-shaped, triangular, or rhomboidal in shape. H.E. Scale bar: 50 μm. (B) There is a neurofilament 68 kDa (NF68)-positive fiber in the subepidermal dermis and it enters the epidermis (arrow). Immunostain with anti-NF68 antibody. Scale bar: 50 μm. (C) Some of the periderm cells (arrowhead) and basal cells (arrow) of the epidermis show faintly positive reaction for nerve growth factor (NGF). No other constituent cells and tissues in the epidermis and the dermis express NGF-positive substances. Immunostain with anti-NGF antibody. Scale bar: 50 μm. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Fourteenth fetal day. Histology and immunohistochemistry. (A, left top) Epidermal and dermal structures are essentially similar to those on the fetal day 13; however, the dermo-epidermal junction (arrow) is distinctly seen. H.E. Scale bar: 50 μm. (Ba, right top) A neurofilament 68 kDa (NF68)-positive fiber extends along the dermo-epidermal junction. Immunostain with anti-NF68 antibody. Scale bar: 50 μm. (Bb, left bottom) Many NF68-positive fibers are seen in the epidermis. Immunostain with anti-NF68 antibody. Scale bar: 50 μm. (C, right bottom) Some of the periderm cells (arrowhead) and basal cells (arrow) of the epidermis showed faintly positive reaction for nerve growth factor (NGF). Immunostain with anti-NGF antibody. Scale bar: 50 μm. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Fifteenth fetal day. Histology and immunohistochemistry. (A) Intermediate cells between the periderm cells and the basal cells of the epidermis appear first in some parts of the epidermis. The basal cells of the epidermis have a short and thick, rectangular nucleus whose long axis is parallel to the skin surface. H.E. Scale bar: 50 μm. (B) There is a distinct trend that all the neurofilament 68 kDa (NF68)-positive fibers decrease obviously in number; however, they are situated at regular space in the possible subpapillary dermis. Immunostain with anti-NF68 antibody. Scale bar: 50 μm. (C) Intermediate cells of the epidermis show strongly positive reaction for nerve growth factor (NGF). Immunostain with anti-NGF antibody. Scale bar: 50 μm. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Sixteenth fetal day. Histology and immunohistochemistry. (A) One to two layers of the intermediate cells appear throughout the epidermis. The basal cells of the epidermis are arranged regularly on the dermo-epidermal junction, and their nuclei are oval or round in shape. In the subepidermal dermis, the small-sized, undifferentiated mesenchymal cells are distributed densely. H.E. Scale bar: 50 μm. (B) The neurofilament 68 kDa (NF68)-positive fibers disappear in the subepidermal dermis and the epidermis, whereas the number of the NF68-positive fibers increases slightly in the possible subpapillary dermis compared with that of the 15th fetal day. Immunostain with anti-NF68 antibody. Scale bar: 50 μm. (C) The intermediate cells are seen as an almost continuous line of the nerve growth factor (NGF)-positive substances in the suprabasal portion of the epidermis. The periderm cells and the basal cells of the epidermis are immunostained negatively with NGF. Immunostain with anti-NGF antibody. Scale bar: 50 μm. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Eighteenth fetal day. Histology and immunohistochemistry. (A) The epidermis consists distinctly of three layers, i.e., basal, spinous, and granular cell layers. The spinous and granular cell layers consist of one to three layers of spinous and granular cells, respectively. No periderm cells are throughout the epidermis any more. H.E. Scale bar: 50 μm. (B) No neurofilament 68 kDa (NF68)-positive fibers are seen in the subepidermal dermis and the epidermis, but in the possible subpapillary dermis the NF68-positive fibers have almost the same mode of the distribution as that of the 16th fetal day. Immunostain with anti-NF68 antibody. Scale bar: 50 μm. (C) Spinous and granular cells of the epidermis show strongly positive reaction, but the basal cells of the epidermis are immunostained negatively with nerve growth factor (NGF). Immunostain with anti-NGF antibody. Scale bar: 50 μm. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Twentieth fetal day. Histology and immunohistochemistry. (A) The horny and granular cell layers of the epidermis are thickened. The horny cell layer shows laminated orthokeratosis. The granular cell layer consists of several layers of the granular cells. Most of the basal cells of the epidermis are cuboidal in shape. There are many developing hair follicles protruding toward the dermis. In the subepidermal dermis, a moderate number of the spindle-shaped, undifferentiated mesenchymal cells are distributed whose long axes are parallel to the skin surface. H.E. Scale bar: 50 μm. (B) A small number of the neurofilament 68 kDa (NF68)-positive fibers (arrowhead) are seen in the subepidermal dermis and few of them (arrow) are in the epidermis. Immunostain with anti-NF68 antibody. Scale bar: 50 μm. (C) The granular and spinous cells of the epidermis show strongly positive reaction; however, not all of the horny cells are immunostained weakly with nerve growth factor (NGF). The basal cells of the epidermis are not immunostained. Immunostain with anti-NGF antibody. Scale bar: 50 μm. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Electron microscopy. (A, left) Thirteenth fetal day. Axoplasm is packed by abundant microtubules (arrowheads) and neurofilaments (arrows); × 48,000. (B, right) Fourteenth fetal day. There is a bundle of immature, grouped axons (asterisk), which is ensheathed incompletely by a probable Schwann cell (S), in the subepidermal dermis. Arrowheads indicate basal lamina of the epidermis; × 7400. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Electron microscopy. Sixteenth fetal day. There are degenerated axons (asterisks), which are neighboring to normal axons (A) × 40,000. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Appearance and disappearance of the cutaneous nerve fibers and expression of nerve growth factor (NGF) of the epidermal cells at different fetal days. There is no relationship between them. Journal of Investigative Dermatology 2004 123, 763-770DOI: (10.1111/j.0022-202X.2004.23434.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions