Milinkovitch Et Al, (2013)- Crocodile Head Scales Are Not Developmental Units but Emerge From...

7/29/2019 Milinkovitch Et Al, (2013)- Crocodile Head Scales Are Not Developmental Units but Emerge From Physical Cracking

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DOI: 10.1126/science.1226265, 78 (2013);339Science

et al.Michel C. Milinkovitchfrom Physical CrackingCrocodile Head Scales Are Not Developmental Units But Emerge

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Evolution, and Climate. B.G.H. also thanks the Marie CurieActions under the Seventh Framework Programme(PIEF-GA-2009-252888). M.B.A. also thanks theSpanish Research Council (CSIC) for support, and S.A.F.thanks the Landes-Offensive zur EntwicklungWissenschaftlich-konomischer Exzellenz program ofHesses Ministry of Higher Education, Research, and theArts. We thank L. Hansen for help with data and reference

compilations. We thank the International Union for Conservationof Nature and Natural Resources for making the amphibianand mammal range data available. Data are archived athttp://macroecology.ku.dk/resources/wallace.

Supplementary Materialswww.sciencemag.org/cgi/content/full/339/6115/74/DC1Materials and Methods

Figs. S1 to S11Tables S1 to S5Appendices S1 and S2References (30729)

1 August 2012; accepted 15 November 201210.1126/science.1228282

Crocodile Head Scales Are NotDevelopmental Units But Emergefrom Physical CrackingMichel C. Milinkovitch,1* Liana Manukyan,1 Adrien Debry,1 Nicolas Di-Po,1 Samuel Martin,2

Daljit Singh,3

Dominique Lambert,4

Matthias Zwicker3

Various lineages of amniotes display keratinized skin appendages (feathers, hairs, and scales) thatdifferentiate in the embryo from genetically controlled developmental units whose spatialorganization is patterned by reaction-diffusion mechanisms (RDMs). We show that, contrary to skinappendages in other amniotes (as well as body scales in crocodiles), face and jaws scales of

crocodiles are random polygonal domains of highly keratinized skin, rather than geneticallycontrolled elements, and emerge from a physical self-organizing stochastic process distinct fromRDMs: cracking of the developing skin in a stress field. We suggest that the rapid growth of thecrocodile embryonic facial and jaw skeleton, combined with the development of a very keratinizedskin, generates the mechanical stress that causes cracking.

Amniotes exhibit a keratinized epidermis

preventing water loss and skin append-

ages that play major roles in thermoregu-

lation, photoprotection, camouflage, behavioral

display, and defense against predators. Whereas

mammals and birds evolved hairs and feathers,

respectively, reptiles developed various types of

scales. Although their developmental processes

share some signaling pathways, it is unclear

whether mammalian hairs, avian feathers and

feet scales, and reptilian scales are homologous

or if some of them evolved convergently (1). In

birds and mammals, a reaction-diffusion mech-

anism (RDM) (2) generates a spatial pattern of

placodes that develop and differentiate into fol-

licular organs with a dermal papilla and cycling

growth of an elongated keratinized epiderm

structure (hairs or feathers) (3). However, scain reptiles do not form true follicles and mig

not develop from placodes (4). Instead, reptil

scales originate in the embryo from regular derm

epidermal elevations (1). Whereas the regu

spatial organization of scales on the largest p

tion of thereptilian body is determined by a RD

additional positional cues are likely involved

the development of the scale plates present

the head of many snakes and lizards. These he

scales form a predictable symmetrical patte

(Fig. 1A) and provide mechanical protection

The face and jaws of crocodilians are cove

by polygonalscales (hereafter called headscale

that are strictly adjoining and nonoverlappibut these polygons are irregular and their spa

distribution seems largely random (Fig. 1

and C). Using high-resolution three-dimensio

(3D) geometry and texture reconstructions (5

1Laboratory of Artificial and Natural Evolution (LANE), Depmentof Genetics and Evolution,University of Geneva, ScieIII, 30, Quai Ernest-Ansermet, 1211 Geneva, Switzerland.Ferme aux Crocodiles, Pierrelatte, France. 3Computer GrapGroup, University of Bern, Switzerland. 4Department of Mematics and Namur Center for Complex Systems, UniversitNamur, Belgium.

*To whom correspondence should be addressed. [email protected]

Fig. 1. Spatial distribu-tion of head scales. (A)Head scalesinmost snakes(here, a corn snake) arepolygons (two upper pan-els) with stereotyped spa-tial distribution(twolowerpanels): left (yellow) andright (red) scale edgesoverlap when reflectedacross the sagittal plane(blue).(B) Polygonal headscales in crocodiles havea largely random spatialdistribution without sym-metrical correspondencebetween left and right.(C) Head scales from dif-ferent individuals havedifferent distributions ofscales sizes and localiza-tions (blue and red edgesfromtop andbottomcroc-odiles, respectively).

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as well as developmental biology techniques,

we show that crocodilians head scales are not

genetically controlled developmental units and

that their spatial patterning is generated through

physical cracking of a living tissue in a stress

field. This phenomenon might not involve any

specific genetic instruction besides those asso-

ciated with cell proliferation and general physical

parameters such as skin stiffness and thickness.

By marking and analyzing various features

directly on 3D models of multiple Nile croco-dile (Crocodylus niloticus) individuals (Fig. 1 and

movie S1), we show that spatial distribution of

head scalesis largely random. First, reflection of

the network of scales edges across the sagittal

plane indicates high variability between the left

and right head pattern (Fig. 1B and fig. S1A).

Second, nonrigid alignment (8) of head geom-

etries from different individuals indicates a

similarly large variability in scale patterns in

terms of polygons sizes and localizations (Fig. 1C

and fig. S1B).

This combination of order and chaos in the

distribution of head scales is reminiscent of the

topological assemblage of soap foams (9, 10).Recent studies used the 2D foam model for

studying self-organizing principles and stochas-

tic processes shaping epithelial topology dur-

ing growth and homeostasis (1113) because the

causal cell-surface mechanics is comparable to

the physics of foam formation (14). Similarly,

the pattern of crocodile head scales could result

from energy minimization of contact surfaces

among genetically determined elements (scales).

However, two other mechanisms could gener-

ate random distributions of polygonal elements:

(i) a RDM patterning the spatial organization of

genetically determined developmental units, as

for mammalian hairs or avian feathers, and (ii)

cracking of a material layer causing its fractureinto adjacent polygonal domains (15).

Although stochastic patterns generated by

these processes share some universal mathe-

matical properties (see supplementary materials),

foams and crack patterns are generated by very

different physical phenomena that may be iden-

tified on the basis of other statistical features.

First, crocodile head scales do not show a good

fit to the area distribution function expected for

foams (fig. S3). Second, a fundamental differ-

ence between foams and crack patterns is that the

latter can exhibit incomplete edges (15), of which

many are observed on the head of crocodiles

(Fig. 2A).Another key feature is the angle among edges

at nodes. In foams, edges are circular arcs in-

tersecting only three at a time with an angle of

120, as imposed by the three instantaneous

equal length-tension force vectors acting a

node. This rule is observed in all types of foam

including animal epithelia (12, 16), althou

the distribution of angles can be widened due

local stress generated by cell division andgrow

On the other hand, crack patterns can gen

ate various angle distributions. Nonhierarchi

cracking arises when fractures propagate sim

taneously (Fig. 2B), and junctions tend to fo

at 120 (17, 18). Furthermore, when a crack fr

splits, or when multiple cracks are nucleatfrom a single point, the junctions among edg

also tend to be 120. Conversely, crack patte

can be hierarchical (17, 19); that is, fractures

formed successively, and propagating cra

will tend to join previous cracks at a 90 ang

Indeed, the local stress perpendicular to a cra

is relaxed and concentrates at the tip of the cra

(explaining its propagation), but the stress co

ponent parallel to the crack is not affected. Hen

as cracks propagate perpendicularly to the

rection of the maximum stress component

secondary crack can turn when it approach

an older one and tends to join it at 90. Sim

larly, if a crack starts on the side of an oldcrack, it will initially tend to propagate a

right angle (17). Multiple examples of 90 co

nections and incomplete edges reorienting th

propagation front are visible on the crocodi

face and jaws (Fig. 2C). We also observe l

dering patterns (17) of paired parallel prim

fractures with perpendicular multiple second

cracks (Fig. 2D) and internal edges connecti

perpendicularly to the border of the netwo

(Fig. 2E). The distribution function of edge a

gles is bimodal in many crocodiles analyz

(fig. S4A), suggesting either that hierarchi

and nonhierarchical cracking processes coex

or that head scale networks undergo a

marationprocess (2022) (see supplementary te

Dome pressure receptors (DPRs) are p

mented round submillimetric sensory orga

(Fig. 2F), distributed on the crocodile face a

jaws, that detect surface pressure waves, allo

ing crocodiles to swiftly orient, even in darkne

toward a prey perturbing the water-air interfa

(23). The dome shape of DPRs is due to a mo

fied epidermis and the presence of a pocket

various cell types in the outermost portion

the dermis (Fig. 2F). We marked the localiz

tion of DPRs on the 3D models of all scann

individuals (orange dots, Fig. 2, G and H). Ma

of the cracks that have stopped their course

so close to a DPR (Fig. 2G and fig. S4C). Giv

that the most frequent cause for fracture arr

is when the crack front meets a heterogeneity

the system (15), it is likely that the modifi

skin thickness and composition at and arou

DPRs constitute such heterogeneities. In

dition, the course of many edges avoids DP

(Fig. 2H and fig. S4C).

The overall distribution of DPRs seems rat

homogeneous except where the density is

creased near the teeth and decreased at the ba

of the jaws and on the top of the face (fig. S

Fig. 2. Signatures of cracking. (A) Many scale edges on crocodiles head areunconnected at one or both ends.(B) Three incomplete cracks interact symmetrically. (C) Edges reorienting (arrows) and connecting with anglesclose to 90. (D) Laddering between parallel primary cracks. (E) 90 network border connections. (F) DPRsare pigmented sensory organs (left) with a modified epidermis (right,section at embryonicstage E70, that is,at70 days of egg incubation) and a pocket of branched nerves (white arrowheads). (G) Incomplete cracksstopping in close proximity to a DPR (orange dots). (H) Crack propagation avoids DPRs.

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Different crocodile individuals differ by as much

as 21% and 48% in their total number of DPRs

and crack edges, respectively. Remarkably, these

two interindividual variations are inversely cor-

related: Crocodiles with fewer DPRs have more

crack edges (fig. S4D). Given that the develop-

ment of DPRs precedes cracking, this correla-

tion suggests that DPRs constrain cracking, as

already implied by Fig. 2, G and H. Despite the

fact that the distributions of cracks and DPRs

both have a strong stochastic component, theconstraining effect of DPRs on cracking is no-

ticeable: The edges tend to travel along the zones

of DPRs lowest local density (fig. S4E).

The archetypal cracking process in physics

is due to shrinkage [through removal of a dif-

fusing quantity, either heat or a liquid (20)] of a

material layer adherent to a nonshrinking sub-

strate (15, 17), such that a stress field builds up

and causes fractures when the stress exceeds a

threshold characteristic of the material. Croc-

odiles have a particularly thick and rigid skin

due to the presence of a highly collagenous

dermis and an epidermis rich in b-keratins (24).

The skin covering their head shows a yet thicker(about 2) and more keratinized epidermis. We

suggest that the rapid growth of the crocodile

embryonic facial and jaw skeleton (relative to the

size of the neurocranium), combined with the

development of a very keratinized skin, gener-

ates the mechanical stress that causes cracking.

Here, it is not the cracking layer that shrinks

but the underlying substrate layer that grows. It

explains that first-order cracks (fig. S6) tend to

traverse the width of the face because the head

is growing longitudinally faster than in other

directions.

In snakes and lizards, scales are develop-

mental units: Each scale differentiates and growsfrom a primordium that can be identified by in

situ hybridization with probes targeting genes

belonging to signaling pathways involved in

early skin appendage development (1, 4). The

large head scales form a predictable pattern

following positional cues, such that the identity

of adult snake head scales can be recognized

while they develop from primordia in the em-

bryo (Fig. 3A). In crocodiles, all postcranial

scales follow that same principle of develop-

ment (Fig. 3B): Spatial distribution of primordia

is established, then each primordium differen-

tiates, first into a symmetrical elevation and sec-

ond as an oriented asymmetrical scale overlapping

with more posterior scales (Fig. 3C).

However, crocodile head scales do not form

from scale primordia or further developmental

stages. Instead, a pattern of DPRs primordia is

generated on the face and jaws: The dome shape

of DPRs has already started to form before

any scale appears (Fig. 3D). Afterward, grooves

progressively appear, propagate, and intercon-

nect (while avoiding DPRs) to form a continuous

network across the developing skin (Fig. 4A).

The process generates polygonal domains of

skin, each containing a random number of DPRs.

Therefore, scales on the face and jaws of croc-

odiles (i) are not serial homologs of scales else-

where on thebody and (ii) are not even genetically

controlleddevelopmental units. Instead, they emerge

from physical cracking.

During a typical cracking process, fractures

are nucleated at the upper surface but quickly

spread downward and affect the whole thick-

ness of the material layer (19). The developing

skin on the crocodiles head similarly reacts to

the stress field as it develops deep groves that

can reach the stiff underlying tissues (Fig. 4B).

Our analyses indicate that cell proliferation in

the epidermis layer is vastly increased in the

deepest region of the skin grooves correspond-

ing to cracks (Fig. 4C), suggesting that a heal-

ing process allows the skin layer to maintain its

continuous covering. The local biological pro-

cess (cell proliferation) might be driven by the

purely physical parameter (mechanical stress) as

follows: In zones of highest stress, local bulging

is nucleated. The local stress component p

pendicular to the bulge is relaxed and conc

trates at its tip, explaining the propagation

both the stress and proliferation maxima (hen

the corresponding propagation of the bulge).

a manner entirely analogous to true physi

cracking, the bulge front would propagate p

pendicularly to the direction of maximum str

components, explaining the topology of the

sulting random polygonal domains of skin. T

role of proliferation reinforces the above sugg

tion that crack patterns in crocodiles might exp

rience maturation(2022), explaining the observ

mixture of hierarchical and nonhierarchical f

tures (see supplementary text and fig. S4A).

We have shown that the irregular polygon

domains of skin on the crocodile face and ja

are produced by cracking, a mechanism tha

distinct from those generating scales on

postcranial portion of the crocodile body,

well as on the body and head of all other re

Fig. 3. Crocodile head scales are not developmental units. (A) In snakes, each body scale (ventrallatero-dorsal, ld) differentiates from a primordium (Shh gene probe for in situ hybridization, cosnake embryo); head scales also develop from primordia, with positional cues determining scidentity (la, labial scales; r, rostral; n, nasal; in, internasal; pf, prefrontal; pro, preocular; so, supraoculpto, postocular). (B) Postcranial scales (zoom on trunk, Ctnnb1 probe) also develop from primordia t

(C) differentiate into symmetrical, then orientedasymmetrical and overlapping, scales. (D) Crocodile hescales never form scale primordia [nor developmental stages shown in (C)] but, instead, developattern of DPRs (one DPR circled with dotted line; dome shape visible at E45) before any scaappears (probe: Ctnnb1).

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tiles. This cracking process is primarily physical.

However, it does not mean that genetically con-

trolled parameters are irrelevant. For example,

although a crack pattern is visible in all croc-

odilian species, spatial distribution varies consid-

erably, possibly because of species-specific skull

geometry and growth but also skin composition

and thickness. Given that these parameters, as

well as cell proliferation, are genetically con-

trolled, the variation of head crack patterns among

crocodilian species is likely due to an interplay

between physically and genetically controlled

param eters . Our stud y suggests that, besides

RDM, a larger set of physical self-organizational

processes contribute to the production of the

enormous diversity of patterns observed in living

systems.

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7. M. Kazhdan, M. Bolitho, H. Hoppe, EurographicsSymposium on Geometry Processing 2006, 61(2006).

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9. D. Weaire, S. Hutzler, The Physics of Foams (Oxford Univ.Press, Oxford, 1999).

10. D. Weaire, N. Rivier, Contemp. Phys. 25, 59 (1984).11. T. Lecuit, P. F. Lenne, Nat. Rev. Mol. Cell Biol. 8, 633

(2007).12. M. C. Gibson, A. B. Patel, R. Nagpal, N. Perrimon, Nature

442, 1038 (2006).13. D. A. W. Thompson, On Growth and Form (Cambridge

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Acknowledgments: This work was supported by the Univeof Geneva, the Swiss National Science Foundation, and thSchmidheiny Foundation. A. Tzika helped with in situs. H.assisted with nonrigid registration. We thank R. Pellet forassistance in mechanics design and A. Roux, M. Gonzalez-GaiB. Chopard, U. Schibler, and anonymous reviewers foruseful comments and suggestions.

Supplementary Materialswww.sciencemag.org/cgi/content/full/science.1226265/DC1Material and MethodsSupplementary TextFigs. S1 to S6Table S1Movie S1References (2533)

18 June 2012; accepted 29 October 2012Published online 29 November 2012;10.1126/science.1226265

Fig. 4. Crocodile headskin cracks during devel-opment. (A) There is nosign of cracking at E45(but DPRs primordia arealready developed, Fig.3D), then primary cracks(arrowheads) appear onthe sides of the upperjaws and progress toward

the top of the face (dottedline).AtE65, primary cracksreached the top of thehead and are followedby secondary cracks inother orientations (ar-rows). (B) Three sequen-tial skin sections alongprimary (pc) and second-ary (sc) cracks (ep, epi-dermis; de, dermis; bo,bone tissue). (C) Antibodyto pan cadherin stains thewholeepidermis, antibodyto proliferating cell nucle-

arantigen(PCNA)indicatesincreased proliferation (ar-rows), and terminal deox-ynucleotidyl transferasemediated deoxyuridinetriphosphate nick end la-beling (TUNEL) assay indi-cates absence of apoptosisin cracks.

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