Supersense: it’s a snap for crocs



Our molecular analyses indicate that small melanin-pigmented integumentary organs (ISOs) in crocodylians express a large repertoire of transduction channels involved in mechano-, thermo-, and chemo-sensory functions. Our electrophysiological analyses confirm that each ISO exhibits a combined sensitivity to mechanical, thermal and chemical stimuli (but not hyper-osmotic salinity), making them remarkable multi-sensorial micro-organs with no equivalent in the sensory systems of other vertebrate lineages. We also show that ISOs all exhibit similar morphologies and modes of development, despite forming at different stages of scale morphogenesis across the body.







Distribution and structure of cutaneous sensory micro-organs in crocodylians



Crocodylians exhibit small melanin-pigmented integumentary organs distributed on virtually all scales (cranial and post-cranial) in gharials and crocodylids, while they are absent from post-cranial scales in alligators and caimans. These organs were initially named Integumentary Sensory Organs (ISOs) then were renamed Dome Pressure Receptors (DPRs) when behavioral and eletrophysiological analyses demonstrated their mechano-sensory abilities [Soares, Nature 2002, 417:241; Leitch et al. J Exp Biol 2012, 215:4217]. Here, we analyzed and compared the structure, innervation, embryonic morphogenesis and sensory function of post-cranial, cranial and lingual sensory organs in two crocodylians species differing by their distribution of ISOs (i.e., cranial and post-cranial, or cranial only): the Nile crocodile (Crocodylus niloticus) and the spectacled caiman (Caiman crocodilus). Our work demonstrates that crocodylians evolved a highly-armored, but very sensitive, skin through the assembly of multiple sensory receptors (diffusely distributed in other amniotes) into discrete micro-organs. The two pictures below indicate the position of ISOs on head scales (left panel) and body scales (right panel) in Nile crocodile. ISOs-like structures are also present on the tongue. Much additional information (including histological analyses, immuno-staining) is available in our publication (see Ref. at the end of this page).







Embryonic development of cranial, post-cranial, and lingual sensory units



We describe the morphogenesis time-course of the skin and associated sensory organs. Scale development is not synchronous across the body of Nile crocodile and alligator embryos. Scale morphogenesis initiates on the tail and dorsum, then on the ventrum, then on the neck and limbs (white arrowheads below indicate the initiation of scale formation on the different body regions). On the body, scale morphogenesis occurs through a conserved mechanism involving the production of symmetrical dermo-epidermal elevations that become progressively asymmetrical depending on their final degree of overlap. In contrast, crocodylians face and jaws scales never form from such developmental stages as they are not genetically-controlled developmental units. Indeed, face and jaws scales emerge from a process entirely analogous to physical cracking of a living tissue in a stress field [Milinkovitch et al. Science 339, 78 (2013)].







The pattern of ISOs on the head is fully formed when head “scales” start to form by cracking of the developing skin. Now, we show here that the formation of ISOs across the body of Nile crocodiles is not synchronized with the process of scale morphogenesis. For instance, the formation of ISOs is clearly visible in the neck scales when they are still symmetrical, while it is observed when scales start to become asymmetrical in the ventral region or when they have basically completed their asymmetrical development in dorsal scales. Finally, we show that ISO formation always involves (including on the jaws and tongue) a local thickening of the epidermis together with condensation of the underlying dermal cells.



Innervation pattern and Multi-sensorial functions of crocodylian ISOs



To compare the innervation pattern of sensory organs in Nile crocodiles and spectacled caimans, we analyzed by immuno-histochemistry the density and distribution of several major neural proteins and neuropeptides within the cutaneous sensory neurons of ISOs. We also explored the nature and distribution of sensory receptors in ISOs. We examined, both with semi-quantitative RT-PCR and whole-mount in situ hybridization (WMISH), the expression patterns of different conserved families of transduction channels in ISO-bearing crocodylian skin tissues. Our RT-PCR on ISO-bearing skin from jaws, dorsum, ventrum, neck, and tongue indicate a similar positive expression of all tested mechano-receptor as well as polymodal chemo- and thermo-receptor channels in crocodiles and caimans. Note that we found significant species-specific differences in the expression pattern of transduction channels responsive to cold temperatures. Our WMISH analyses indicate that all transduction channels identified by RT-PCR are specifically expressed in developing ISOs during embryogenesis and nowhere else in the skin. This strongly suggests that ISOs might be involved in multiple functions, in addition to previously identified surface wave detection.

To test our hypothesis, we performed in-vivo extracellular electrophysiological recordings from individual ISOs in Nile crocodiles and spectacled caimans to assess the sensory modalities of post-cranial and cranial ISOs using different local stimuli that naturally occur in their environment (changes of pressure, temperature, salinity and pH). We confirmed that ISOs are able to detect mechanical stimuli and further demonstrate that all types of ISOs additionally respond to both warm and cold temperatures as well as to both increase and decrease of pH and to TRPV1-3 agonists. Note that we did not detect any electrophysiological response of Nile crocodile and caiman ISOs to hyper-tonic concentrations (equivalent to seawater). This suggests that the abilities of some crocodylians to discriminate salinity is not associated to ISOs but to other sensory organs (probably in the oral cavity) that remain to be identified.



Conclusions



Our study shows that crocodylian ISOs all exhibit similar morphologies and modes of development during embryogenesis, despite that they differ in distribution, density and timing of formation among scales across the body. Most importantly, we show that ISOs exhibit a combined sensitivity to mechanical stimuli, cold and warm temperatures, and pH variations, making them remarkable multi-sensorial micro-organs with no equivalent in the sensory systems of other vertebrate lineages. Altogether, this study indicates that the ancestral vertebrate diffused sensory system of the skin was transformed in the crocodylian lineages into an array of discrete multi-sensory micro-organs innerved by multiple pools of sensory neurons. This discretization of skin sensory expression islands is unique among vertebrate and allowed crocodylians crocodilians develop a highly-armored, but very sensitive, skin.







Publications

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