SPRR1 Gene Induction and Barrier Formation Occur as Coordinated Moving Fronts in Terminally Differentiating Epithelia  Damian Marshall, Matthew J. Hardman, Carolyn Byrne  Journal of Investigative Dermatology  Volume 114, Issue 5, Pages 967-975 (May 2000) DOI: 10.1046/j.1523-1747.2000.00955.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Permeability change on murine fetal skin showing formation of the skin barrier (Hardman et al. 1998). Black areas (negative by permeability assay) represent pre-barrier skin and white areas (positive by permeability assay) represent post-barrier epidermis: (A–D) increasing gestational age; arrows show moving front; asterisks show initiation sites. Journal of Investigative Dermatology 2000 114, 967-975DOI: (10.1046/j.1523-1747.2000.00955.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Diagrammatic representation of (A) tongue development and (B) palate development. (A) The floor of the pharynx is derived from the first, second, and third branchial arches (1–3). Two lateral swellings and a median swelling arise from the first arch. These will later expand and fuse to form the oral portion of the tongue (at approximately E11.5–12 of mouse development). Tissue from the third branchial arch overgrows the second arch and fuses with the oral portion of the tongue to form the pharyngeal region. (B) Tissue from the premaxilla forms the primary palate, whereas secondary palate derives from fusion of palatal shelves at approximately E14.5 of mouse development. Journal of Investigative Dermatology 2000 114, 967-975DOI: (10.1046/j.1523-1747.2000.00955.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 (A) Permeability assays applied to fetal dorsal tongue at increasing gestational age (i–viii) and in adult (ix): (i) absence of barrier at approximately 16 d 0 h gestation (E16/0); (ii) barrier formation initiates at sites on the terminal sulcus (asterisks), and then moves along the midline (median sulcus, white arrow in iii); permeability change encircles the intermolar eminence (IE, iv), and then fronts of permeability change move to the periphery of the tongue (arrows, v and vi) and anteriorly over the intermolar eminence (arrow, vi); (vii) by approximately E17/9 the assay indicates complete permeability change except for a remnant on the intermolar eminence. Papillae characteristic of adult tongue (viii, ix) stain purple. IE, intermolar eminence. (B) Barrier formation in fetal hard palate: (i) pre-barrier palate at approximately E17/0; (ii–v) increasing gestational age; (ii) fusion line appears to act as an initiation region (palatine raphe, asterisks show change). Note that the anterior part of the primary palate changes before the secondary palate. (iii) Permeability change moves down the midline fusion (white arrow); (iv) fronts of permeability change move to the periphery (arrows, iv). Change is complete by E18/5. Note that buccal epithelium (arrowheads, v) remain blue (negative by permeability assay). (C) Diagrammatic interpretation of pattern change on tongue dorsal surface (i) and palate (ii). Fusion lines are shown by red dotted lines. First initiation sites activated are shown as dark blue stars and later sites are at fusion lines shown by light blue dotted lines. (i) Anterior (oral or first-arch-derived) tongue develops by fusion of the median tongue bud and the two lateral tongue buds. Lateral tongue buds fuse at the median sulcus. Oral tongue also fuses with pharyngeal tongue (mainly third-branchial-arch-derived) and the fusion line is called the terminal sulcus. (ii) Palate develops by fusion of primary palate (from the intermaxillary part of the maxilla) and secondary palatal shelves. The secondary palatal fusion line is called the palatine raphe. Journal of Investigative Dermatology 2000 114, 967-975DOI: (10.1046/j.1523-1747.2000.00955.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 TEWL falls coincident with permeability barrier formation on the dorsal surface of the tongue. AT E16/2 TEWL is high indicating lack of permeability barrier in tongue. A statistically significant (p = 0.0293) fall in TEWL has occurred by E16/12 coincident with barrier formation and corresponding to acquisition of barrier levels approaching adult levels. By E17/2 TEWL has fallen to a level indistinguishable from adult tongue. TEWL values obtained from the tongue, however, are substantially higher than those obtained from adult skin, demonstrating that the tongue permeability barrier never reaches comparable competence to the skin barrier. TEWL values represent the mean of at least six data sets. Error bars: ± s.e.m. Journal of Investigative Dermatology 2000 114, 967-975DOI: (10.1046/j.1523-1747.2000.00955.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 (A)–(D) Comparison of adult and fetal (E16/8) oral epithelia. Light micrographs of adult dorsal tongue (A) and adult secondary palate (C) show characteristic prominent stratum corneum (SC). In contrast, fetal tongue (B) and fetal palate (D) lack stratum corneum and are greatly reduced in thickness. (E)–(L) Electron micrographs demonstrating barrier formation on tongue and palate. Specific morphologic changes accompany barrier formation on the dorsal surface of a transitional tongue (E, inset; approximate locations of electron micrographs E–H marked). Pre-barrier epithelium from the edge of the tongue (E) is thin, shows nucleated surface cells, and has yet to form papillae. Further towards the barrier front the epithelium increases in thickness accompanied by a coincident increase in papillae maturity (F). Immediately before the barrier front crosses the epithelium (G) the uppermost cells flatten considerably and electron-dense granules can be seen aggregating at the cell periphery (F, G, arrowhead). At the barrier front (G) there is a sudden change in morphology, characterized by the appearance of a single thin, anucleate, electron-dense cell (G, arrow) directly below a nucleated upper layer. At the center of the tongue more flattened electron-dense cells appear (H, arrow) and the upper nucleated cell appears to be degrading. The ends of these electron-dense cells protrude into the epithelia at right angles to the surface and taper to a point. Analogous morphologic change accompanies barrier formation in a transitional secondary palate (I, inset; locations of electron micrographs I–L marked). At the edge of the palate the epithelium is very thin with rudimentary stratification (I) Further towards the center of the palate, near the outer edge of the rugae, the epithelium increases in thickness and stratification becomes well defined (J). At the barrier front, aggregates appear at the cell periphery (K, arrowhead), and then flattened electron-dense cells appear (K, L, arrow) as in tongue. At the center of the palate the number of electron-dense cells again increases. Palatal cells are considerably thinner and straighter, however, than those in tongue. Scale bar: 24 μm (A–D), 2 μm (E–H), 3.6 μm (I–K), and 4.5 μm (L). Journal of Investigative Dermatology 2000 114, 967-975DOI: (10.1046/j.1523-1747.2000.00955.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Comparison of SPRR1 gene induction and barrier formation patterns. (A) Whole tongues were cut sagittally and half was assayed for barrier formation by permeability staining and half assayed for SPRR1 gene induction by wholemount in situ hybridization. Tongues were then realigned for comparison. (I) Early expression (E16/0) of SPRR1 gene occurs in barrier pattern prior to barrier formation on the tongue dorsal surface. (II) As barrier forms (E16/10) there is an apparent downregulation of SPRR1 gene expression due to exclusion of probe/antibody by the emerging barrier; however, expression is still present in regions where barrier has formed, as shown clearly along the dissection line (arrowheads, II, III, and IV). (III) Several hours later (E16/16) as the barrier acquisition front propagates across the tongue dorsal surface, barrier-associated riboprobe exclusion also increases. Note that even though the barrier moving front has almost reached the outer edges of the tongue there is still no SPRR1 expression in the intermolar eminence. (IV) Detail from (II) SPRR1 gene expression is prominent along the dissection line demonstrating that the gene is active (arrowheads). Note the absence of SPRR1 gene expression along the dissection line of the intermolar eminence, demonstrating delayed gene induction in this region. (V, VI, VII) Tissue sections from regions indicated in (ii) and (iv); e, stratified epithelia; d, dermis. (V) SPRR1 gene is expressed in outer layers of stratified epithelia of tissue that has formed a barrier, despite a negative response by wholemount in situ hybridization due to probe exclusion; (VI) the front of SPRR1 gene induction at the junction of the intermolar eminence and surrounding tissue; (VII) section showing SPRR1 gene expression from tissue that has not formed a barrier. Here, SPRR1 gene expression is detected by both wholemount and section analysis. Sense probe (not shown) gives a uniformly negative response. Scale bar: 37 μm (V–VII). (B) Multiple moving fronts appear to cross the tongue dorsal surface epithelia in sequentially older tongues. Wholemount in situ hybridization results are shown in the upper panel and a diagrammatic interpretation of the results in the lower panels (C). Initially expression of SPRR1a gene on E16/0 tongue dorsal surface occurs in a typical barrier pattern prior to barrier formation. As the expression of SPRR1 reaches the outer edges of the tongue dorsal surface by E16/4 barrier starts to form. Moving fronts of barrier formation then begin to propagate from the midline at E16/10, preventing entry of probe/antibody into the underlying epithelia. Note the extreme similarity between the pattern of gene expression at E16/0 and the pattern of barrier formation at E16/10. By E16/16 the fronts of SPRR1 gene expression and barrier formation are almost identical; however, expression remains absent from the intermolar eminence until E17/0, with barrier formation following at around E17/5. (C) Diagrammatic representation of the waves of moving fronts on tongue dorsal surface epithelia representing the developmental stages in (B). Upper row shows expression patterns of the SPRR1 gene (yellow) in sequentially older tongues. Lower row demonstrates both gene expression (yellow) and barrier formation pattern (pink) and shows that the results produced experimentally are due to barrier-associated probe exclusion in wholemount in situ hybridization studies. (D) SPRR1 gene expression in hard palate (I, III, V) demonstrated by wholemount in situ hybridization resembles the barrier formation pattern (II, IV, VI). Probe/antibody exclusion also occurs where epithelial barrier forms (arrowhead in V). Asterisks in (I)–(IV) show initiation sites; arrows in (III) show the direction of moving fronts. Journal of Investigative Dermatology 2000 114, 967-975DOI: (10.1046/j.1523-1747.2000.00955.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions