Chemical and mechanical patterning of tortoise skin scales occur in different regions of the head

Abstract

Vertebrate skin appendages are diverse micro-organs such as scales, feathers, and hair. These units typically develop from placodes, whose spatial patterning involves conserved chemical reaction-diffusion dynamics. Crocodile head scales are a spectacular exception to this paradigm, as they instead arise from a mechanically dominated process of compressive folding driven by constrained skin growth. Here, we reveal that chemical versus mechanical processes pattern tortoise scales in different regions of their head. Indeed, we show that placode-derived scales emerge across the peripheral head surfaces while remaining absent from the central dorsal region where scales subsequently form through a mechanical folding process. Using light sheet microscopy, we build a three-dimensional mechanical model that qualitatively recapitulates the diversity of scale patterns observed in this central head region in different tortoise species. Overall, our analyses indicate that mechanical head-scale patterning likely arose before the divergence between Testudinata and Archosauria, and was subsequently lost in birds.

Keywords: Biological sciences; Evolutionary biology; Zoology.

Graphical abstract

Figure 1

The diversity of tortoise head scale patterns (A) 3D reconstructions of juvenile tortoise heads from structured light scanning microscopy, including the sulcata tortoise (Centrochelys sulcata), the marginated tortoise (Testudo marginata), and the Greek tortoise (Testudo graeca). These meshes reveal that the central dorsal head surfaces exhibit signatures of mechanical patterning, including unjoined scale edges (yellow arrowheads).^, (B) Embryonic development of sulcata tortoise head scales. At E50, scales are absent. By E55, polygonal domains propagate across the peripheral dorsal head surface and lateral regions of the head (second column, white arrows). By E60, the head is mostly covered with polygonal scales, except for the central dorsal surface, where unjoined scale edges first become visible (yellow arrowhead). These unjoined scale edges continue to propagate from E65 to E70, giving rise to irregular polygons. By E70, the entire central dorsal head also exhibits fine-scale 3D geometry (inset).

Figure 2

The development of sulcata tortoise head scales (A and B) WMISH reveals expression of the classic placode markers Ctnnb1 (A) and Shh (B), localized to peripheral head scale primordia, from E55 to E60. Cryosections of these samples reveal the expression of Shh (B, bottom row) in a nested subregion of Ctnnb1-expressing epidermal cells (A, bottom row). By E65, cryosections of WMISH samples reveal a dense, keratinous, and undulating epidermis in the peripheral placode-derived region, lacking the local expression of Ctnnb1 and Shh (A and B, bottom right panels) because patterning is completed. Conversely, placode-associated gene expression is never observed in the central dorsal head skin. (C) We use nanoindentation to examine changes in the skin surface stiffness of the embryonic sulcata tortoise head during the propagation of unjoined scale edges (C, left panel). We observed a rapid increase in effective Young’s modulus from E55 to E70 (C, right panel), revealing a substantial increase in skin surface stiffness in the central dorsal head surface. Mean values (±SD) are shown for each biological replicate.

Figure 3

Building a mechanical model of tortoise central dorsal head scale patterning (A) We use LSFM to capture the precise tissue layer geometry of embryonic sulcata tortoise heads, from E55 until E70. Surface reconstructions from nuclear staining (with TO-PRO-3 Iodide; top row) show the emergence of unjoined scale edges (yellow arrowheads) and asymmetric scale domains on the dorsal head surface. Optical sections (middle row) reveal the rapid thickening and keratinization of the densely packed epidermis from E55 to E70, as well as the spatial distribution of proliferating cells (labeled with EdU) in the epidermis and dermis. Alizarin red staining reveals the progressive ossification of the skull (bottom row), revealing that the onset of skin surface patterning in the central dorsal head surface only occurs after the near-complete development of the skull at E65. (B) Left panel: we build a 3D finite-element numerical growth model integrating the tissue layers segmented from the LSFM data. Right panel: proliferating cell densities indicates somewhat homogeneous growth within the dermis and the epidermis (see also Figure S3), such that the numerical model assumes homogeneous growth within each layer but allows for different values between them (Table 1).

Figure 4

Our mechanical model recapitulates tortoise central dorsal head scale patterning (A–C) Variation of elastic and growth parameters (Table 1) allows us to qualitatively recapitulate the normal patterning of the central dorsal head region observed in sulcata tortoises ((A), see also Video S1), Greek tortoises ((B), see also Video S2) and marginated tortoises ((C), see also Video S3).

Figure 5

The developmental diversity of reptilian head scale patterning Reptilian head scales can arise in accordance with one of three processes: (A) head scales can emerge from individual placodes patterned via paradigmatic chemical RD as observed in snakes (here, the corn snake, Pantherophis guttatus); (B) head scales can also arise from a purely mechanical process of compressive folding driven by frustrated skin growth, as observed in crocodilians (here, the Nile crocodile, Crocodylus niloticus); or (C) head scales can emerge from sequential chemical and mechanical processes occurring in different regions of the head, as observed in tortoises (here, a Galápagos giant tortoise, Chelonoidis niger). In this latter process, the peripheral scales first emerge from RD-patterned placodes before compressive folding gives rise to skin surface patterning in the central dorsal head surface. (D) As mechanical patterning processes contribute to the head scale patterning of both crocodiles^, and tortoises (this study), mechanical compressive (growth-driven) head scale patterning is likely a synapomorphic trait that arose before the divergence between Archosaurs and Testudinata, and was subsequently lost in birds (Aves).