Research ArticleEVOLUTIONARY BIOLOGY

The anatomical placode in reptile scale morphogenesis indicates shared ancestry among skin appendages in amniotes

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Science Advances  24 Jun 2016:
Vol. 2, no. 6, e1600708
DOI: 10.1126/sciadv.1600708

Figures

  • Fig. 1 Development of epidermal scales during reptilian embryogenesis.

    (A) Hematoxylin and eosin (H&E) staining of skin sections from different body regions (indicated with red arrows on the top insets with lateral views of corresponding embryos) of C. niloticus (crocodile; top row), P.vitticeps (lizard; two middle rows), and P. guttatus (snake; bottom row) embryos at various developmental stages [indicated as embryonic days (E) after oviposition]. White arrowheads indicate the anatomical placode. Scale bars, 100 μm. (B) Anatomical placodes in C. niloticus (left panels), P.vitticeps (middle panels), and P. guttatus (right panels) embryos. For each species, the whole-embryo WMISH with Sonic hedgehog (Shh) is shown (left panel) as well as, from top to bottom, high magnification of H&E-stained placode sections (white arrowheads indicate placode columnar cells), immunohistochemistry with PCNA (proliferation marker; epidermal-dermal junction indicated by dashed white lines), and parasagittal cryosections of placodes after Shh or β-catenin (Ctnnb1) WMISH. Bmp4 is also shown for lizard. Red double-headed arrows indicate the body region processed for sectioning.

  • Fig. 2 Macropatterning of developing scales in reptiles.

    (A and B) WMISH with Ctnnb1 in C. niloticus and P. vitticeps embryos at various developmental stages. Arrowheads indicate the initiation sites of scale tracts and arrows indicate the directions of scale tracts. Colors correspond to different tracts schematically represented in the right panels (dots, initiation sites; arrows, directions of development). (C) WMISH with Shh in P. guttatus embryos at various developmental stages. Arrowheads with white borders indicate tract initiation sites, and arrowheads with black borders indicate the boundaries of Shh expression at different developmental stages, showing the different anteroposterior (a/p) and ventrodorsal (v/d) gradients (see schematic in the right panel).

  • Fig. 3 Characterization of mutant scaleless P. vitticeps lizards.

    (A) Dorsal views of adult wild-type (WT) and scaleless (Sca) P. vitticeps lizards. The white arrowhead indicates the presence of large lateral spines in the WT. (B) Ventral views of WT and Sca adult males showing the absence of femoral pores (arrowheads) in mutant lizards. (C) Micro x-ray computed tomography scan virtual sections of the skull (left) and magnified views of the autopod (right) of WT and Sca dragons at birth. White frames indicate the position of the pleurodont regenerating teeth, and double-headed arrows show the relative sizes of claws. (D) Diagram of WT (EDAWT) and mutant scaleless (EDASca) active EDA proteins. The conserved collagen and TNF domains are shown as black and gray boxes, respectively. The most conserved TNF motif [17 amino acids (aa) in WT] is shown in red. The mutant EDA protein has an in-frame deletion of 14 amino acids, as shown by the alignment of EDA protein sequences from mouse, chicken, and WT and Sca P. vitticeps (lower panel). Black numbers represent amino acid position. (E) Upper panel: diagram showing the genomic structure (from exon 7 to 8) of the P. vitticeps Eda gene. Intron length and splice donor (gt) and acceptor (ag) sites are indicated. Blue arrows show the positions of primers used for reverse transcription polymerase chain reaction (RT-PCR) analyses. In the scaleless mutant genome, a transposon of 5.7 kb starting with an alternative splice donor site is inserted in the 3′ end of exon 7, thus leading to an alternative splicing (red dashed lines) of the mutant EdaSca gene in comparison to the splicing of the EdaWT gene (black dashed lines). Lower panels: RT-PCR analysis (g, on genomic DNA; c, on skin cDNA) of WT and Sca animals using the indicated primer combinations. (F) Top row: H&E staining of skin sections from dorsal and lateral body regions of adult WT and scaleless dragons. Middle row: immunofluorescent staining of α-keratins (α-k) and β-keratins (β-k) or laminin (lam; arrowhead shows convoluted basal membrane) in dorsal skin of adult WT and scaleless animals. Bottom row: Toluidine blue (TB) staining of dorsal skin sections and scanning electron microscopy (SEM) images of skin molts from adult WT and scaleless lizards. is, interscale region; os, outer scale region. Scale bars, 50 μm. (G) H&E staining of dorsal skin sections of scaleless P. vitticeps embryos at various developmental stages (indicated as embryonic days after oviposition); red arrows in the top insets indicate the locations of skin sections on lateral views of the corresponding embryos. Scale bars, 100 μm.

  • Fig. 4 Absence of anatomical placodes in scaleless P. vitticeps skin.

    (A to C) WMISH showing the expression of early markers of epidermal appendage development in WT and Sca bearded dragon embryos at various indicated developmental stages: (A) Shh; (B) Ctnnb1; and (C) Edar (left), Eda (center), and Bmp4 (right). Left panels show the WMISH signal on the lateral skin region, and right panels show parasagittal cryosections of the corresponding regions. Insets show high magnifications of the staining and indicate the presence/absence of placode formation in WT and mutant skin, respectively.

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