Development 100, 171-177 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

171

Tissue polarity in an insect segment: denticle patterns resemble

spontaneously forming fibroblast patterns

K. NUBLER-JUNG

Institutfiir Biologie I (Zoologie) der Albert-Ludwigs-Universitat Freiburg, Alberistrasse 21a, D-7800 Freiburg FRG

Summary

The insect integument displays planar tissue polarityin the uniform posterior orientation of denticles andbristles. How do cell polarities become uniformlyoriented in the plane of the epidermal sheet? We havealready shown that it is possible to disturb uniformdenticle orientation in abdominal segments of Dysder-cus (Niibler-Jung, 1987). Here I report that abnor-mally oriented denticles tend to form small arrayswith uniform orientation. Adjacent arrays with diver-gent orientations realize a small repertory of charac-teristic pattern elements. We obtain these patternelements by orthogonal transformation of patternelements that form spontaneously in confluent fibro-blast cultures, which rely on autonomous cell behav-iour, and which later simplify into patterns predictedby specific boundary conditions (Elsdale & Wasoff,

1976); the only additional parameter required isplanar cell polarity. The abnormal patterns in Dys-dercus may thus also form spontaneously and mayalso rely on autonomous cell behaviour. The normalpattern is predicted by the parallel segment bound-aries.

I propose that the characteristic pattern elements inthe larval epidermis may arise because elongatedepidermal cells tend to arrange in parallel arrays andto orient in the same direction. The normal posteriororientation of cell polarities may result from orientingcues provided by the anterior and by the posteriorintersegmental regions.

Key words: insect, epidermis, tissue polarity, cellpolarity, pattern formation, denticle patterns, fibroblastpatterns, Dysdercus intermedius.

Introduction

Biological patterns are often large when compared tothe size of individual cells. On one hand, disturbancesin the supracellular pattern are often repaired locally,i.e. by cells in and around the disturbed regions.Pattern repair thus seems to call for supracellularmechanisms that inform individual cells where theyare and how they should behave (e.g. Wolpert, 1971;Meinhardt & Gierer, 1980). On the other hand, thereis increasing evidence that purely mechanical con-straints such as boundary geometry and certain ratherunspecific cell properties can produce very specificsupracellular patterns in vitro (e.g. Harris, Stopak &Warner, 1984) and in vivo (e.g. Oster, Murray &Harris, 1983). Here I propose that the supracellularpolarity pattern displayed in an insect segment mayalso rely on local interactions between adjacent cells.Together with the boundary conditions set by theintersegmental region they may stabilize the normalpattern.

The insect integument has long been a modelsystem for the analysis of pattern repair mechanisms(e.g. Wigglesworth, 1959). Earlier authors had con-cluded that a supracellular cue graded in antero-posterior (in leg and wing proximodistal) directioncontrols the sequence of differentiated cells (Bohn,1965; Locke, 1967). In addition, such a gradient couldprovide orientation and thus could introduce polaritynot only in single cells but also in the tissue as a whole(Stumpf, 1966; Lawrence, 1966; Lawrence, Crick &Munro, 1972). Subsequent experiments indicatedthat cell adhesion is graded in anteroposterior direc-tion and may be involved in maintaining the normalsequence of differentiated cells (Bohn, 1974; Law-rence, 1974; Niibler-Jung, 1974, 1977). However,graded cell adhesion does not easily explain how apolarity pattern is being controlled. Moreover, thepolarity patterns observed after grafting (Locke,1966; Nardi & Kafatos, 1976a,b) and after woundhealing (Locke, 1967; Lawrence et at. 1972; Niibler-Jung, Bonitz & Sonnenschein, 1987) do not always

172 K. Nubler-Jung

conform to the gradient model. I therefore startedlooking for mechanisms independent of a gradientthat could orient the polarity pattern in an insectsegment.

Earlier I proposed that divergent surface proper-ties of the anterior and of the posterior intersegmen-tal regions could induce and orient cell polarities inthe segment (Sander & Nubler-Jung, 1981). Morerecent experiments indicate that insect epidermalcells can establish planar polarity independently of acommon orientation with surrounding cells (Nubler-Jung, 1987). Here I report that the insect integumentcan form complex supracellular polarity patterns.These polarity patterns are formally equivalent to thedynamic and selfsimplifying planar patterns that arisespontaneously by local cell interactions in confluentfibroblast cultures (Elsdale & Wasoff, 1976). I pro-pose that the polarity pattern in an insect integumentmay also arise by local cell interactions which arebased on a tendency of epidermal cells to orient in thesame direction. If so, a supracellular graded cue maynot be necessary to control the polarity pattern in aninsect segment.

Materials and methods

Cotton bugs (Dysdercus intermedius Dist., Heteroptera)were reared as described in Nubler-Jung (1977), except thatthey were kept at 30 ± 1°C. Fifth (= last) instar larvae wereinjected with about 0-4 fig colchicine. The adult cuticle wasobserved after ecdysis. For further details of procedure seeNubler-Jung (1987).

Results and interpretation

The insect integument consists of a monolayer ofepidermal cells and the overlying cuticle. A newcuticle is secreted before each moult.

In adult cotton bugs (Dysdercus intermedius) theventral part of the abdomen carries cuticular denticlesand bristles which point posteriorly. In some regions(5th and 6th segment and medial part of 2nd and 3rdsegment) each cell forms a bristle or a denticle. Weassume that bristle or denticle orientation reflects theorientation of planar polarity in the underlying epi-dermal cell at the time of cuticle secretion. Uniformorientation of cuticular protrusions indicates a supra-cellular tissue polarity.

Fig. 1 shows the normal polarity pattern of uni-formly oriented bristles and denticles in the adultcuticle. After a single colchicine injection into mid-fifth (= last) instar larvae, the adult cuticle displaysfewer bristles than normal, probably because colchi-cine impairs differential cell divisions. In addition,

Fig. 1. Imaginal cuticle of the 5th abdominal sternite.Each epidermal cell secretes a denticle (small structure)or a bristle (elongated structure) and thereby expressesplanar cell polarity. Planar tissue polarity is indicated byuniform posterior orientation of cuticular protrusions.Top, anterior of animal; phase contrast; bar, 10(im.

Fig. 2. Four types of pattern elements (broad bands)form around a discontinuity where denticles with oppositeorientation lie side by side (thin lines). A loop (A) and aji (B) around the ends of a discontinuity line. (C) Awhorl around a point discontinuity. (D) A 'double-^1

around a short discontinuity line. Phase contrast; bar,lOfim.

the denticles (and bristles) in the adult cuticle point invarious directions (Niibler-Jung, 1987). Yet, the den-ticles are hardly ever oriented at random. Rather,they form smaller or larger areas of uniform thoughabnormal orientations.

Tracing the major denticle orientations reveals thatabnormal patterns are characterized by essentiallytwo types of pattern elements which can be describedas a loop and a n. These pattern elements form

Polarity patterns in an insect segment 173

around the ends of a linear discontinuity with tangen-tially opposing denticles (Fig. 2A,B). We also findwhorls that can be considered as two apposing loops,and 'double ris' formed by two apposing single Jt's(Fig. 2C,D). Large areas with abnormal denticleorientations display pairs of pattern elements aroundthe ends of a single discontinuity line in all fourcombinations (Fig. 3); small areas display mainlypairs of one loop and one n (Fig. 4).

Fig. 3. Denticle pattern in the adult (2nd sternite) 6 days after injection of colchicine into last larval instar. (A) Largearea with abnormal denticle orientations. Bristles are scarce. (B) Discontinuity lines (thin lines) separate denticles withtangentially opposing orientations. The broad bands tracing uniform denticle orientations are continuous and formessentially two pattern elements, a loop or a n. A whorl and a 'double-.Tr' can be considered as two apposing loops andtwo apposing n"s respectively. Top, anterior of animal; phase contrast; bar, 10^m.

174 K. Nubler-Jung

Fig. 4. Denticle pattern in the adult (5th sternite) 6 daysafter colchicine injection into last larval instar. Withcolchicine epidermal cells in the 5th segment tend to formmultidenticulate denticles and only a few bristles.Discontinuity lines (thin lines) separate denticles withtangentially opposing orientations. The broad bandstracing uniform denticle orientations are continuous andform pairs of one loop and one n. Top, anterior ofanimal; phase contrast; bar, 10[im.

Animals injected with colchicine as mid-fifth instarlarvae produce abnormal cuticle patterns while ani-mals injected earlier produce normal patterns(Nubler-Jung, 1987). This may indicate that cellpolarities become initially disoriented by colchicinebut later recover and adopt the normal posteriororientation.

Discussion

(A) Analogies between denticle patterns and fibroblastpatterns

The pattern elements observed in the integument ofDysdercus can be obtained by a formal transform-ation of pattern elements that arise spontaneously ina confluent monolayer of fibroblasts in long-termculture. Migrating single fibroblasts tend to adhereside by side and then adopt an elongated bipolarspindle form (Elsdale, 1972; Elsdale & Bard, 1972).The confluent culture thus consists of a patchwork ofnumerous independently formed parallel arrays. Ar-rays with divergent orientations merge by smooth

I

D

Fig. 5. Four types of pattern elements. Thin lines:arrangements of elongated fibroblasts as arisespontaneously in confluent fibroblast cultures (Elsdale &Wasoff, 1976). Thick line with arrows: main orientation ofpolarity as resulting from fibroblast pattern by orthogonaltransformation and introduction of polarity.(A) Fibroblasts form a 'half-sun', its transformation givesa loop. (B) Fibroblasts form a triradius, itstransformation gives a it. (C) Fibroblasts form a 'whole-sun', its transformation gives a whorl. (D) Fibroblastsform a 'four-radius', its transformation gives a 'double-.^'.

changes in orientation around essentially two typesof point discontinuities (Elsdale & Wasoff, 1976).Orthogonal transformation with introduction of po-larity will transform these discontinuities into thepattern elements observed in Dysdercus (Fig. 5, com-pare with Fig. 2).

The formal similarity between fibroblast patternsand denticle patterns may indicate that fibroblastsand insect epidermal cells share specific cell proper-ties that allow for this sort of pattern formation.

Fibroblasts tend to elongate along a boundary andalong another fibroblast (Elsdale, 1972; Elsdale &Bard, 1972). Between a pair of parallel boundariesthe resulting pattern is a continuum of parallelelongated cells (Elsdale & Wasoff, 1976). Its orthog-onal transformation with introduction of polarity willgive an array of uniformly oriented denticles suchas characterizes the pattern in a normal segment(Fig. 6).

The normal insect epidermis forms an array ofmediolaterally elongated cells, at least in the apical

Polarity patterns in an insect segment 175

A

Orthogonaltransformationand polarity

L

V V VV V

v v vv vV V V

v v

B

Fig. 6. In a small field with parallel boundariesfibroblasts form a continuum of a single parallel array(A); its orthogonal transformation with introduction ofpolarity gives a uniform polarity pattern, e.g. uniformlyoriented denticles (B).

Fig. 7. Mediolateral elongation at apical cell regionduring secretion of adult epicuticle (6-5-day-old fifthinstar larva). Middle segment region; anterior (dark) cellscontain red pigment, posterior (light) cells contain whitepigment. For adult cuticle pattern see Fig. 1. Top,anterior of animal; bar, 10jum.

region and during the beginning of cuticle secretion,i.e. where and when cuticular protrusions are beingconstructed (Fig. 7; see also Wigglesworth, 1973).The similarity between denticle patterns and fibro-blast patterns may thus result because insect epider-mal cells also tend to elongate along each other andalong the parallel segment boundaries.

The cellular equivalent for the orthogonal trans-formation of fibroblast patterns into denticle patternsmay thus be that insect epidermal cells tend to orienttheir denticles and bristles perpendicular to their longaxes (Fig. 7; see also Figs 6-10 in Lawrence, 1969).Interestingly, denticles secreted during wound heal-ing also point perpendicular to the axis of putativecell elongation and therefore in abnormal directionswith reference to the segment borders (Nubler-Jungetal. 1987).

(B) Uniform polarity pattern in a segment may relyon local cell interactions and on global constraintsElsdale & Wasoff (1976) showed that the fibroblastpattern in a unitary, close-packed field is subject to

constraints that can be described and quantified onlyin terms of the whole field. Such 'global constraints'arise because fibroblasts tend to align along a bound-ary, for instance a scratch on the plastic substrate.Together with the 'local constraints' (i.e. the tend-ency of fibroblasts to elongate along each other) theglobal constraints predict the final arrangement ofcells. For example, in a field with two parallelboundaries the spontaneously forming pattern willsimplify into a single field of parallel arrays (Fig. 6). Ipropose that likewise the normal polarity pattern inan insect segment is the result of local and globalconstraints.

The local constraints could derive from an inherenttendency of neighbouring cells to orient in the samedirection. I have proposed before that localized butpotentially mobile cell surface molecules might trans-mit orientation between adjacent cells (Sander &Nubler-Jung, 1981; see also Held, Duarte & Derakh-shanian, 1986). Mediolateral elongation of insectepidermal cells during cuticle secretion (Fig. 7) mayreflect an increased membrane contact between cellsabout to transmit orientation in anteroposteriordirection.

The global constraints may derive from the segmentboundaries. Here mediolateral elongation of epider-mal cells is even more pronounced than in thesegment (Lawrence & Green, 1975), possibly becausecells with divergent surface properties meet (Nubler-Jung, 1979). The segment margins may thus define aboundary along which other segment cells tend toalign and therefore will have their anteroposterioraxis defined. The specific posterior orientation ofdenticles may be determined by the different sign (+or —) of orienting properties of the anterior andposterior intersegmental regions (Piepho, 1955ft;Nubler-Jung, 1979). Whether these orienting proper-ties are based on diffusible molecules (Lawrence et al.1972) or on cell-bound structures (Sander & Nubler-Jung, 1981) remains to be investigated.

With this explanation local cell interactions wouldprovide for uniform orientation of epidermal cellpolarities (tissue polarity) while the segment bound-aries would align cell polarities along an anteropos-terior axis.

In confluent fibroblast cultures the elongated cellscontinue to adopt parallel configurations. As a result,the supracellular pattern simplifies by mutual elimi-nation of unlike discontinuities (Elsdale & Wasoff,1976). In the insect epidermis apposed tissues withdivergent polarity tend to adopt uniform orientations(e.g. Piepho, 1955a; Lawrence, 1974; Lawrence &Shelton, 1975; Nubler-Jung & Grau, 1987). Abnor-mal polarity patterns in the insect epidermis may thusalso simplify by mutual elimination of unlike patternelements (Fig. 8).

176 K. Nubler-Jung

Fig. 8. Hypothetic mutual elimination of discontinuities.When divergent polarities tend to adopt uniformorientations then two opposing loops and two opposing;r's (A) will shorten into one whorl and one double-^respectively (B). Elimination of the separation betweenwhorl and double-^r results in a pair of one loop and oneJI (C) which will approach one another and finally willannihilate each other to give a pattern of uniformorientation. Lines with arrows indicate predominantdenticle orientations.

Simplification of abnormal polarity patterns intothe normal pattern predicted by the 'global con-straints' is compatible with the observation thatanimals moulting more than 7 days after colchicineinjection again display normal cuticle patterns(Nubler-Jung, 1987).

I do not exclude that insect epidermal cells canorient along some graded cue, e.g. graded cell ad-hesion (Locke & Huie, 1981a,b; Nardi & Magee-Adams, 1986) or a graded morphogen concentration(Lawrence et al. 1972). However, since (1) normaland abnormal polarity patterns in Dysdercus can beexplained on the basis of specific modes of next-neighbour cell interactions, and since (2) insect epi-dermal cells can orient independently of a putativegradient (Locke, 1966; Lawrence etal. 1972; Nardi &Kafatos, 1916a,b; Nubler-Jung, 1987; Nubler-Jung etal. 1987), I propose that a supracellular graded cuemay not be necessary to control the polarity patternin an insect segment.

It is certainly tempting to assume that duringembryogenesis the polarity pattern in an insect seg-ment is being laid down by the cellular mechanismsthat appear to maintain the larval polarity pattern.Yet, these mechanisms often do not predict theorientation in an intercalary regenerate (Bohn, 1965;French, 1978; Lawrence et al. 1972) while a gradientmodel does. This warns us that de novo generation ofthe polarity pattern (in embryogenesis or regener-ation) and its subsequent control during postembry-onic development may rely, at least in part, ondifferent mechanisms.

I am most grateful to Dr M. G. Vicker for drawing myattention to the Elsdale and Wasoff paper. I also thankProf. Dr K. Sander and Dr P. A. Lawrence for very usefulsuggestions. The help of Frau M. Scherer with typing themanuscript and of Frau B. Mardini with production of thepictures is gratefully acknowledged.

References

BOHN, H. (1965). Analyse der Regenerationsfahigkeit derInsektenextremitat durch Amputations- undTransplantationsversuche an Larven der afrikanischenSchabe (Leucophaea maderae Fabr.). II. Mitteilung.Achsendetermination. Wilhelm Roux' Arch. EntwMech.Org. 156, 449-503.

BOHN, H. (1974). Pattern reconstitution in abdominalsegment of Leucophaea maderae (Blattaria). Nature,Lond. 248, 608.

ELSDALE, T. (1972). Pattern formation in fibroblastcultures, an inherently precise morphogenetic process.In Towards a Theoretical Biology, vol. 4 (ed. G. H.Waddington), pp. 95-108. Edinburgh: University Press.

ELSDALE, T. & BARD, J. (1972). Cellular interactions inmass cultures of human diploid fibroblasts. Nature,Lond. 236, 152-155.

ELSDALE, T. & WASOFF, F. (1976). Fibroblast culturesand dermatoglyphics: the topology of two planarpatterns. Wilhelm Roux' Arch, devl Biol. 180, 121-147.

FRENCH, V. (1978). Intercalary regeneration around thecircumference of the cockroach leg. J. Embryol. exp.Morph. 47, 53-84.

HARRIS, A. K., STOPAK, D. & WARNER, P. (1984).Generation of spatially periodic patterns by amechanical instability: a mechanical alternative to theTuring model. J. Embryol. exp. Morph. 80, 1-20.

HELD, C. I., DUARTE, C. M. & DERAKHSHANIAN, K.(1986). Extra tarsal joints and abnormal cuticularpolarities in various mutants of Drosophilamelanogaster. Wilhelm Roux' Arch, devl Biol. 195,145-157.

LAWRENCE, P. A. (1966). Gradients in the insect segment:the orientation of hairs in the milkweed bug Oncopeltusfasciatus. J. exp. Biol. 44, 607-620.

LAWRENCE, P. A. (1969). Cellular differentiation andpattern formation during metamorphosis of themilkweed bug Oncopeltus. Devl Biol. 19, 12-40.

Polarity patterns in an insect segment 177

LAWRENCE, P. A. (1974). Cell movement during patternregulation in Oncopeltus. Nature, Lond. 248, 609-610.

LAWRENCE, P. A., CRICK, F. H. C. & MUNRO, M. (1972).A gradient of positional information in an insect,Rhodnius. J. Cell Sci. 11, 815-853.

LAWRENCE, P. A. & GREEN, S. M. (1975). The anatomyof a compartment border: the intersegmental boundaryin Oncopeltus. J. Cell Biol. 65, 373-382.

LAWRENCE, P. A. & SHELTON, P. M. J. (1975). Thedetermination of polarity in the developing insectretina. J. Embryol. exp. Morph. 33, 471-486.

LOCKE, M. (1966). The cuticular pattern in an insect: thebehaviour of grafts in segmented appendages. J. InsectPhysiol. 12, 397-402.

LOCKE, M. (1967). The development of patterns in theintegument of insects. Adv. Morph. 6, 3-88.

LOCKE, M. & HUIE, P. (1981a). Epidermal feet in pupalsegment morphogenesis. Tissue & Cell 13, 787-803.

LOCKE, M. & HUIE, P. (19816). Epidermal feet in insectmorphogenesis. Nature, Lond. 293, 733-735.

MEINHARDT, H. & GIERER, A. (1980). Generation andregeneration of sequence of structures duringmorphogenesis. J. theor. Biol. 85, 429-450.

NARDI, J. B. & KAFATOS, F. C. (1976a). Polarity and

gradients in lepidopteran wing epidermis. I. Changes ingraft polarity, form, and cell density accompanyingtranspositions and reorientations. J. Embryol. exp.Morph. 36, 469-487.

NARDI, J. B. & KAFATOS, F. C. (1976ft). Polarity and

gradients in lepidopteran wing epidermis. II. Thedifferential adhesiveness model: gradient of a non-diffusible cell surface parameter. J. Embryol. exp.Morph. 36, 489-512.

NARDI, J. B. & MAGEE-ADAMS, S. M. (1986). Formation

of scale spacing patterns in a moth wing. I. Epithelialfeet may mediate cell rearrangement. Devi Biol. 116,278-290.

NUBLER-JUNG, K. (1974). Cell migration during patternreconstitution in the insect segment {Dysdercusintermedius Dist., Heteroptera). Nature, Lond. 248,610-611.

NUBLER-JUNG, K. (1977). Pattern stability in the insectsegment. I. Pattern reconstitution by intercalaryregeneration and cell sorting in Dysdercus intermedius(Dist.). Wilhelm Roux' Arch, devl Biol. 183, 17-40.

NUBLER-JUNG, K. (1979). Pattern stability in the insectsegment. II. The intersegmental region. Wilhelm Roux'Arch, devl Biol. 186, 211-233.

NUBLER-JUNG, K. (1987). Insect epidermis: Disturbancesof supracellular tissue polarity does not prevent theexpression of cell polarity. Wilhelm Roux' Arch, devlBiol. (in press).

NUBLER-JUNG, K., BONITZ, R. & SONNENSCHEIN, M.(1987). Cell polarity during wound healing in an insectepidermis. Development 100, 00-00.

NUBLER-JUNG, K. & GRAU, V. (1987). Pattern control ininsect segments: superimposed features of the patternmay be subject to different control mechanisms.Wilhelm Roux' Arch, devl Biol. (in press).

OSTER, G. F., MURRAY, J. D. & HARRIS, A. K. (1983).

Mechanical aspects of mesenchymal morphogenesis. /.Embryol. exp. Morph. 78, 83-125.

PIEPHO, H. (1955a). Uber die Ausrichtung derSchuppenbalge und Schuppen am Schmetterlingsrumpf.Naturwissenschaften 42, 22.

PIEPHO, H. (19556). Uber die polare Orientierung derBalge und Schuppen auf dem Schmetterlingsrumpf.Biol. Zbl 74, 467-474.

SANDER, K. & NUBLER-JUNG, K. (1981). Polarity andgradients in insect development. In International CellBiology 1980/1981 (ed. A. G. Schweiger), pp. 497-506.Berlin, Heidelberg: Springer.

STUMPF, H. (1966). Uber gefallabhangige Bildungen desInsektensegmentes. J. Insect Physiol. 12, 610-617.

WIGGLESWORTH, V. B. (1959). The Control of Growth andForm: A Study of the Epidermal Cell in an Insect.Cornell University Press.

WIGGLESWORTH, V. B. (1973). The role of the epidermalcells in moulding the surface pattern of the cuticle inRhodnius (Hemiptera). J. Cell Sci. 12, 683-705.

WOLPERT, L. (1971). Positional information and patternformation. Curr. Top. devl Biol. 6, 183-224.

(Accepted 30 January 1987)