An ancient Turing-like patterning mechanism regulates skin denticle development in sharks

RD simulation and gene expression analyzes suggest that a Turing-like system underlies shark body denticle patterning

We first investigated the morphogenetic patterning of shark denticles. Two rows of dorsal denticle placodes are visible at stage 32 of development [~80 days postfertilization (dpf)] (Fig. 1A) (42), preceding the emergence of denticles (Fig. 1, C, D, and F). Compared to body denticles, dorsal denticles are larger and broader and do not have distinct ridges associated with hydrodynamic drag reduction (Fig. 1, D to F) (5). An example of a dentine denture can be used to determine dentine dentistry. Patterns were generated from a row of dorsal dorsal dentin initiators (Fig. 1K), from which waves of activatory and inhibitory morphogens were radiated according to predefined values ​​(Fig. 1L and Table S1, see Materials and Methods for further details). Spots formed in rows adjacent and parallel to the initiator row. On reaching a steady state, initiator spots of greater shape than newly formed spots (Fig. 1M), reflecting squamation of the shark (Fig. 1, D to J). This model provides a Turing-like system for controlling denticle patterning in sharks.

To compare the pattern of shark denticles and chick feathers, we examined the expression of β-catenin (β-cat), an early regulator of chick epithelial placode signaling (Fig. 2 and Fig. S1) (43). The chicken embryo expresses a dorsolateral stripe of β-cat at embryonic day 6 (E6) (Fig. 2, C and D). This stripe becomes compartmentalized into individual feather placodes at E7 (Fig. 2, G and H), which trigger the emergence of adjacent, parallel placode rows (Fig. 2, K and L) (18). The shark lateral line expresses β-cat at stage 31 (~ 70 dpf), shortly before denticle patterning begins (Fig. 2, A and B). A continuous stripe of expression was observed in the shark; however, two dorsolateral rows of denticle placodes appeared simultaneously at stage 32 (~ 80 dpf), expressing β-cat (Fig. 2, E and F). These rows become parallel to the lateral line (Fig. 2, A to F). The smaller body denticle placodes 32 (~ 100 dpf) (Fig. 2, I and J). Dorsal dorsal shark may be acting as initiation lines, triggering the emergence of surrounding units in a Turing-like mechanism comparable to feather patterning. Having noted this similarity between shark and chick epithelial appendage patterning, we have discussed the expression of genes underlying a putative Turing-like patterning system in the shark.

Using in situ hybridization (ISH), we sought to identify the potential activators and inhibitors of this Turing-like patterning system. As a result of their importance during feather patterning (18), and their expression was analyzed throughout the shark (Fig. 3 and Fig. S1). At stage 31 (~ 70 dpf), dorsal denticle placodes were not detected (Fig. S2), although β-cat expression labeled development of the lateral line sensory system (Fig. 2, A and B). By early stage 32 (~ 80 dpf), two dorsolateral rows of denticle placodes were visible, expressing the known activators of feather patterning, FGF4 and shh, more the inhibitor bmp4 (Fig. 3, A to C) (18, 42). Similar to feather patterning, bmp4 was expressed in placards rather than the interplacode regions, suggesting that its inhibitory action is indirect (18). The mesenchymal marker of feather bud development, FGF3was also expressed in dorsal denticle rows (Fig. 3D) (44), along with the runt domain transcription factor runx2 (Fig. 3E), which is associated with FGF signaling throughout mammalian tooth morphogenesis and mineralization of other vertebrate skeletal elements (Fig.45–47). An anterior to posterior dorsal denture development was noted.

Later in developmental stage 32 (~ 100 dpf), body denticle placodes become visible in rows adjacent and parallel to dorsal denticle rows. Body denticles extend throughout the ventral trunk and eventually propagate to the entire flank and ventral surface. We understand that there are multiple initiation sites (48), which are important for the extension of denticle patterning to the extremities, such as the paired pectoral purposes. Redeployment of the same suite of dentine dorsal dentistry (Figure 3, F to O). ISH section revealed that shh was expressed in the denticle epithelium, while FGF4, bmp4, and runx2 were expressed in the underlying mesenchyma (Fig. 3, P to R and T). The expression of FGF3 was noted in both the epithelium and mesenchyma (Fig. 3S). Overall, these results revealed extensive conservation of RD-related gene expression between denticle and feather patterning (18, 43, 49).

RD-related genes are functionally conserved during the patterning of shark body denticles

To verify the functional conservation of the genes during the denticle patterning, we undertook the small-molecule gene pathway inhibition experiments. Embryos were treated with FGF receptor inhibitor SU5402 (50) or dimethyl sulfoxide (DMSO) as a control. Beads were implanted beneath the epithelium in stage 31 embryos (~ 75 dpf), adjacent to rows of dorsal emerging denticle primordia (Fig. 4A). Development then continued before the genetic and phenotypic effects of treatment were examined at various time points.

First, ISH for RD-related genes was undertaken 5 days posttreatment (dpt). Localized inhibition of shh and bmp4 The present invention has been observed in dorsal dentine placodes treated with SU5402 beads, where DMSO beads (Fig. 4, C to J, and Figs., S3 and S4, A to D) are used. We propose that inhibition of FGF signaling disrupted a conserved activator-inhibitor feedback system between fgf4, shh, and bmp4, which similarly mediates feather patterning (Fig. 4B) (18). Furthermore, we observed down-regulation of sprouty 2 (Spry2) expression (Figure 4, K to N). ace Spry2 is a downstream transcriptional readout of FGF signaling (51This supports the idea that SU5402 treatment leads to FGF inhibition in this system. Sections of whole-mount ISH samples revealed stunted development of denticle primordia (Fig. 4, C to N, and Fig. S4), suggesting that inhibition of FGF signaling during early morphogenesis is sufficient to restrict dorsal denticle growth. As dorsal denticles develop in anterior to posterior gradient, the treatment effect was strongest in the undergoing early morphogenesis at the time of beading, rather than merely the units closest to the bead (Fig. 4, C to N). For example, in Fig. 4K, the bead of the anterior to units with reduced gene expression, as the units closest to the bead are more advanced in their development. Posterior units undergoing early morphogenesis (demarked with a black arrowhead) were affected by the treatment. Growth of the embryo may also affect proximity to the area of ​​inhibition. These results suggest that there is a significant role in the management of shark denticle patterning, with FGF signaling playing an important activatory role.

Next, we examined the effect of the bead implants at 25 dpt, the stage at which smaller body dentures (~ 100 dpf). Using ISH, we visualized FGF4 expression to examine how the disruption of dental dorsal denture subsequent patterning (Fig. 4, O and P, and Fig. S4, E and F). Dorsal denticle primordia failed to undergo morphogenesis following FGF inhibition, resulting in a gap in the row. This gap has become infilled by smaller denticidal denticles (Fig. 4O), which may be used as an adjunct to dorsal denticles. In contrast, control samples displayed in a complete row of dorsal denticles (Fig. 4P). Alizarin red staining of SU5402 beaded samples fixed at 50 and 75 dpt revealed that this pattern was maintained throughout, with smaller, mineralized body denticles occupying the gaps in the dorsal denticle rows (Fig. 4, Q and R, and Figs. S6). Next, we look at this pattern of response with an RD system. Therefore, we simulated the RD model (Fig. 1, K to M) with a unit missing from the initiator row (Fig. 4S) to mimic the functional experiment. The model output boron similarity to the pattern following bead implantation, with smaller units occupying the gap resulting from the star initiator (Fig. 4, T and U). These results provide further evidence that Turing-like systems controls shark dentition patterning, as the model response remains robust following experimental manipulation.

Retuning the RD model can explain the diversity of denticle patterning

Having found evidence for Turing-like denticle patterning in the catshark, we sought to examine the role of this system in other elasmobranch species. Among elasmobranchs, denticles of different densities, with most dense sharks. Comparatively, the dentition coverage of the thornback skate (Raja clavata) and the little skate (Leucoraja erinacea) is enhanced (Fig. 5, A to F). This article presents the results of this study of inhibitors of morphogenesis in the RD model to predict this diversity in elasmobranch denticle density.

Model parameters were initially set to result in a catshark-like denticle pattern (Figs 1, K to M, and 5, D and G). The inhibitor's constitutive degradation rate (d_v) and maximum net production rate (G_max) were then decreased, while its diffusion coefficient (D_v) was increased (table S1). Initiator spots were enlarged and spaced further apart to reflect the dorsal row of the skate (Fig. 5E). This model is comparable to the thornback skate (Fig. 5, E and H). Next, the activator's constitutive production ratec_u) was decreased (table S1). This further reduces the density of coverage, giving rise to similar patterning to the little skate (Fig. 5, F to I). It is worth noting that there are many alternative methods of measurement (Figure 5, G to I), which are more importantly9). Overall, these results demonstrate that simple alterations to the parameters of the RD model can give rise to a wide diversity of patterns of outcomes comparable to those seen in extant elasmobranch species. The plasticity of this system can be seen in the vast spectrum of vertebrate epithelial appendage patterns.