Development 99, 255-259 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

255

Haemocytes secrete basement membrane components in embryonic

locusts

ELDON E. BALL1, HEINZ GERT de COUET1, PEGGY L. HORN2 and JOAN M. A. QUINN1

1 Department of Neurobiology, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra City,ACT 2601, Australia1 Applied Science, Canberra College of Advanced Education, Belconnen, ACT 2616, Australia

Summary

Several monoclonal antibodies raised against a glyco-protein-enriched fraction of adult muscle membranesof Locusta migratoria selectively stain particles withinhaemocytes and basement membrane in developinglocust embryos. Haemocytes containing immuno-reactive particles are found associated with areas

where basement membrane is being laid down. Theunderlying ectoderm does not show immunoreactivity.We conclude that haemocytes contribute to basementmembrane formation in embryonic locusts.

Key words: Locusta migratoria, haemocyte, basementmembrane, monoclonal antibody.

Introduction

There is a long-standing controversy as to whetherhaemocytes secrete basement membrane componentsin insects. Lazarenko (1925) was the first to suggestthat they do, and Wigglesworth (1933, 1956, 1973,1979) has published a series of papers presentingincreasingly convincing evidence for such a role inRhodnius. Ashhurst, however, who has done con-siderable research on insect haemocytes, has writtenseveral recent reviews (1979,1982,1985) in which shefinds the arguments for participation of haemocytes inbasement membrane formation unconvincing. Gupta(1985) lists other papers relevant to the controversyand has summarized the evidence for the two oppos-ing points of view.

In the insect literature the term 'basement mem-brane' has been used for extracellular layers ofconnective tissue both with and without formedelements such as fibrils. In an attempt to arrive at amore uniform usage Ashhurst (1979, p. 326) hasdefined a basement membrane as 'a thin amorphouslayer, between 50 and 80 nm thick, which is foundunder the basal surface of the epithelial cells andaround muscle cells'. The layer of material that wehere refer to as basement membrane fits this descrip-tion except that it is usually thicker than 80 nm.

We here present immunocytochemical and ultra-structural evidence that haemocytes do contribute

directly to basement membrane formation in theembryonic locust, Locusta migratoria.

Materials and methods

A membrane fraction from the metathoracic femoralmuscles of adult Locusta migratoria was prepared by sucrosegradient centrifugation and integral membrane componentswere solubUized by 1 % Triton X-100. A glycoprotein-enriched fraction was obtained by affinity chromatogTaphyover a Concanavalin A-sepharose column. Material thuspurified was injected into BALB-c mice for production ofmonoclonal antibodies (Koehler & Milstein, 1975). Hy-bridomas were cloned once by limiting dilution. For screen-ing of hybridoma culture supernatants whole embryos,staged according to the criteria of Bentley, Keshishian,Shankland & Toroian-Raymond (1979), were dissected freeof yolk and fixed in 2% paraformaldehyde for approxi-mately 20min. The supernatant solutions were thenscreened on these whole-mount embryos, using techniquesdescribed in Ball, Ho & Goodman (1985), at 5 % intervalsfrom 30 to 60 % of embryonic development. Immunoreac-tivity was detected using secondary antibodies labelled witheither fluorescein isothiocyanate (FTTC) or horseradishperoxida§e (HRP). Control embryos were routinely runwith an antibody of known specificity. The monoclonalantibodies were also tested on frozen sections of adultmetathoracic femur.

Embryos were fixed for light and electron microscopy for1-2 h on ice in a mixture of 2% paraformaldehyde, 2-5 %glutaraldehyde and 0-0025 % CaCl2 in Millonig's phosphate

256 E. E. Ball, H. G. de Couet, P. L. Horn and J. M. A. Quinn

ect

Fig. 1. Whole mount of a 30 % embryo of Locusta migratoria in which the basement membrane lining the pleuropodia,which are organs lying behind the metathoracic legs, stains intensely with FTTC (arrows). Scattered haemocytes (out offocus) are also stained. The embryo has been opened in the dorsal midline (bright longitudinal strips) to facilitatepenetration of the antibodies into the coelomic space. Bar, 100 fan.Fig. 2. In the metathoracic leg of a 40 % whole-mount embyro many haemocytes, containing HRP-labelled darklystaining granules, are associated with a dark-staining basement membrane (arrows). Nomarski optics, ect, ectoderm.Bar, 30 /zm.Fig. 3. In the prothoracic leg of a 35 % whole-mount embryo haemocytes containing immunoreactive particles arepresent and the basement membrane has begun to stain with FITC. Bar, 30 am.Fig. 4. Three haemocytes containing FTTC-staining particles are here shown in association with the basement membranelining the ectoderm of the antenna in a 55% whole-mount embryo, ect, ectoderm. Bar, 10/an.Fig. 5. All of the haemocytes in the pleuropodia of a 40 % whole-mount embryo contain HRP-stained immunoreactivegranules. The basement membrane is also stained. Normarski optics, ect, ectoderm. Bar, 10//m.

Haemocytes secrete basement membrane in locust embryos 257

Fig. 6. Semithin transverse section of a pleuropodium. Three of the four haemocytes in its lumen (arrows) are attachedto the basement membrane lining the ectoderm. Bar, 20 fan.Fig. 7. Electron micrograph of a haemocyte in contact with the ectoderm (ect) of a pleuropodium at 42 % of embryonicdevelopment. Note abundant basement membrane (bm) between the haemocyte and the ectoderm and the similarity inappearance between one class of intracellular structure within the haemocyte (large arrowheads) and the basementmembrane. An example of a second class of inclusion which might be immunoreactive is marked with a small arrow.Bar, l^an.Fig. 8. Basement membrane (bm) apparently being laid down between a haemocyte (h) and the underlying ectoderm(ect). Putative immunoreactive material is enclosed within a cisterna of rough endoplasmic reticulum (large arrowhead).A small vesicle can be seen apparently emptying by exocytosis from the haemocyte onto the basement membrane (smallarrowhead), m, mitochondrion. Bar, 0-5 nm.Fig. 9. A large vesicle, containing many smaller vesicles, apparently emerging from a haemocyte (h) into the spacewhere basement membrane is being laid down adjacent to the ectoderm (ect). Bar, 0-25 ^m.

258 E. E. Ball, H. G. de Couet, P. L. Horn and J. M. A. Quinn

buffer with osmolarity adjusted by addition of 1-2%D-glucose (Bate, 1976 and personal communication). Theywere then rinsed in buffer, postfixed on ice for 1 h in 1 %OsO4 in Millonig's buffer, rinsed, stained in 1 % uranylacetate in distilled H2O for 1 h, rinsed in distilled H2O,dehydrated through an ethanol series, and embedded inAraldite resin. Sections for light and electron microscopywere cut using glass knives and stained with toluidine blueor uranyl acetate and lead citrate, respectively.

Results

Four hybridoma cell lines that produced monoclonalantibodies (MAb) specific for particles within haemo-cytes and for basement membrane (Figs 1-5) ofembryonic locusts were isolated. In addition to theirembryonic specificity the antibodies also strongly andspecifically bound to the margins of muscle fibres(presumably to basement membrane) and nerve (pre-sumably to neural lamella) in frozen sections of adultfemoral tissue, providing further evidence that theantigens are localized in connective tissue.

At 30% of embryonic development two of themonoclonal antibodies (HM1B3 and HM2C9) stainonly the inside surface of the pleuropodia and a fewhaemocytes scattered throughout the coelomic cavity(Fig. 1), while MAb HM3A12 stains only haemo-cytes. At this same stage MAb Co3H12 stains bothhaemocytes and adjacent areas of basement mem-brane which are now appearing on the inner wall ofthe ectoderm.

By 40 % of embryonic development immuno-reactive haemocytes are abundant and many arelocated against the inner wall of the ectoderm wherebasement membrane is now staining more intensely.The association occurs throughout the body(Figs 2-5) but we chose to study it ultrastructurally inthe pleuropodia (embryonic organs located posteriorto the metathoracic legs) for three reasons. First,judging by immunoreactivity, the amount of base-ment membrane lining the pleuropodia appears to begreater than anywhere else in the embryo at 40-45 %of embryonic development. Second, haemocytes areabundant within the pleuropodia during this sameinterval, and third, the haemocytes there can beviewed clearly using differential interference contrastoptics and all appear to contain immunoreactivegranules (Fig. 5).

Because the pleuropodia appear to contain auniform population of haemocytes at this stage wewere able to examine thin sections with the assurancethat any cells found in the lumen of the pleuropodiawould contain immunoreactive granules. Fig. 6 showsa transverse section of a pleuropodium with haemo-cytes in its lumen. As shown in Fig. 7, such cellscontain only two types of intracellular structures that

appear to be of the correct size and distribution tocorrespond to the immunoreactive particles seen inwhole-mount preparations. The more common ofthese inclusions (Fig. 7) is located within cisternae ofrough endoplasmic reticulum (Fig. 8) and appearsidentical to the basement membrane in morphologi-cal appearance and staining properties. The othertype of inclusion stains more darkly and containsgranular matrix material (Fig. 7). Fig. 8 shows a smallvesicle apparently emptying by exocytosis from ahaemocyte into the area where basement membraneis being laid down between the haemocyte and theectoderm. In Fig. 9 a large vesicle, containing manysmaller vesicles, is shown apparently emerging from ahaemocyte into the area of basement membranedeposition.

Discussion

Several investigators have applied histochemicalmethods to study the composition and developmentof the extracellular matrix in insects. Although ourantibodies are presumably more specific than theperiodic acid-Schiff (PAS) stain used by Wiggles-worth (1956) our findings are basically similar to his(1956, 1973, 1979): i.e. (a) haemocytes are foundadjacent to the basement membrane at times andlocations where it is apparently being laid down,(b) particles within the haemocytes stain the same asthe basement membrane, and (c) the haemocytes canbe seen apparently releasing material onto areaswhere basement membrane is forming.

Ashhurst (personal communication) has pointedout that histochemical studies on adult locust haemo-cytes (Costin, 1975) failed to detect any of thesubstances that are present in the connective tissuematrix surrounding the ejaculatory duct of the adultlocust. Although we have not biochemically charac-terized the antigens against which our antibodieswere raised it is likely from the way they wereprepared that they represent glycoconjugates thatare commonly found in basement membranes ofboth adult and embryonic tissue. Detergents wereemployed throughout the staining procedures toensure that the antibodies penetrated cell mem-branes, but the only intracellular sites exhibitingimmunoreactivity were the particles within the em-bryonic haemocytes. No binding of antibodies wasobserved in the ectoderm and it thus appears that thisspecific component of the basement membrane isexclusively produced by the haemocytes. It is, how-ever, quite possible either that this class of haemo-cytes or the particles within them are only found inembryonic locusts.

Ashhurst (personal communication) has read ourpaper and concludes that ' . . . either the antibodies are

Haemocytes secrete basement membrane in locust embryos 259

recognizing the same epitope on different moleculesor that they are recognizing an epitope on a moleculesecreted by the blood cell which is being transferredto the underlying epithelial cell, via the basementmembrane'. In answer to the first point, the epitope ispresent on molecules in haemocytes and basementmembrane but not in the epithelial cells, which, asidefrom the haemocytes, would be the other Likelysource of immunoreactive material in the basementmembrane. This combination of coincidences ap-pears to us unlikely, especially in view of the fact that,judged by differences in the time and pattern of onsetof staining by the monoclonal antibodies, we areprobably dealing with more than one immunoreactiveepitope. The evidence against Ashhurst's secondsuggestion is that we have never seen any immuno-reactivity within the epithelial cells.

We conclude that although much of the basementmembrane is clearly secreted by adjacent epithelialcells (Ashhurst, 1979, 1982, 1985) the haemocytes domake a significant direct contribution to at leastcertain areas of basement membrane during embry-onic development of Locusta migratoria.

Although Dr Doreen Ashhurst does not agree with ourconclusions we are grateful for her comments, which haveresulted in a better paper. We thank Aleksandra Plazinskafor help in raising the monoclonal antibodies.

References

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{Accepted 15 October 1986)