J. CeU Set. *8, 309-327 (i977) 309Printed in Great Britain © Company of Biologists Limited 1077

THE ABDOMINAL EPIDERMIS OF THE BLOWFLY

CALLIPHORA ERYTHROCEPHALA

II. IMAGINAL HISTOBLASTS DURING LARVAL LIFE

MURRAY J. PEARSON*School of Biological Sciences, University of Sussex, Falmer, Brighton,Sussex, England

SUMMARY

Abdominal histoblasts in Calliphora (Diptera) are situated within the larval epidermis, andsecrete larval cuticle throughout the 3 larval instars. An optical and electron-microscopic studyof histoblasts and epidermal sensilla suggests that the latter have been previously confused withhistoblast Anlagen in the first instar.

A steady rate of division until the mid-third instar leads to Anlagen growth, which is notseen in larval Drosophila. After a relatively quiescent period, division begins again, at a higherrate, at the time of pupariation (at the time when division first begins in Drosophila). Duringprepuparial larval life, the ventral, dorsal, and postero-dorsaJ Anlagen increase from approxi-mately 6, 8 and 4 cells to about 350, 500 and 150 cells respectively.

At the time of the second (puparial) mitotic phase there is also extensive reorganization ofcellular fine structure in which large secondary lysosomes play an important part. The endo-plasmic reticulum and Golgi subsequently present a different profile. At the same timenumerous chromatic droplets appear in the rapidly dividing histoblast Anlagen. This divisionand the appearance of chromatic droplets are shown (and the subcellular reorganization issuggested) to depend upon the hormonal factors which are responsible for pupariation.

INTRODUCTION

Throughout the course of hypertrophy, and concomitant polyteny, of abdominalepidermal cells in Calliphora larvae (Pearson, 1974) another epidermal population ofsmall imaginal cells remains diploid. These histoblasts are organized in discretegroups - the imaginal Anlagen - in each of the first 7 abdominal segments (Bautz,1971). The last abdominal segment carries 3 genital disks, as in Musca (Diibendorfer,1971).

The cuticular secretory activity of abdominal histoblasts, which function as larvalepidermal cells during the third larval instar (Pearson, 1972), shows that these imaginalcells do not constitute typical imaginal disks; they share neither the histologicalorganization, nor the developmental status of 'embryonic' undifferentiated cellswhich is characteristic of the disks of head, thorax, and genitalia (Ursprung &Nothiger, 1972). The properties of abdominal histoblasts suggest instead a differentsignificance in the biology of dipteran metamorphosis, as well as different developmentalproblems (Garcia-Bellido & Merriam, 1971; Pearson, 1977; Lawrence, Green &

• Author's address: 'Vittles' Coffee House, 21 Gardner Street, Brighton, Sussex, England.

310 M. J. Pearson

Johnston, 1977). Bautz (1974), however, has taken issue with this contention, claimingthat in first instar larvae pedunculate disks may be seen in abdominal sections.

In order to clarify the matter, the histological and fine-structural organization, andthe mitotic behaviour of abdominal histoblasts have been studied right through thelarval life of Calliphora erythrocephala. In addition, this description shows howreorganization of histoblasts precedes their invasion of the general abdominal epidermisat metamorphosis; and in particular leads to an interesting study of secondarylysosomal activity in the redirection of cell function (Pearson, in preparation).

METHODS

Larvae were grown in roughly synchronous cultures under the constant conditions at 25 °Cdescribed by Pearson (1974).

Histological preparation for light microscopy

Whole mounts were prepared by fixing the epidermis in distended form. Larvae weredecapitated and internal organs removed. The decapitated larva was then slipped over the endof a suitably drawn pipette and held by forceps while inflated by Ringers and then Carnoy'sfluid. For optimal fixation, a flow of firstly the inflating Ringers and then, immediately following,the fixative is directed into the larva and out round the neck of the pipette. The operation wasperformed in Ringers. The inflated epidermis was kept in Carnoy's for 7-10 min, then trans-ferred to 70 % ethanol, cut down the mid ventral line, and pinned out for dissecting away thesegmental and intersegmental muscles. Histoblast Anlagen were located in relation to thedorso-ventral muscles on either side. Preparations were taken to water, hydrolysed in N HC1and stained in Feulgen. The fifth abdominal segment was photographed in all cases. Photo-graphy of whole mounts employed phase-contrast optics.

For examination of sectioned material, hypodermal specimens were prepared as for electronmicroscopy (below) and semithin sections stained with methylene blue. Material was alsoexamined in paraffin sections stained with haemalum and eosin.

Electron microscopy

Larvae of the first 2 instars were fixed by inflation with cold 2-2 % glutaraldehyde (TAAB)with the addition of 200 mia D-glucose in 0-05 M sodium cacodylate buffer adjusted to pH 7-0.After a 30-min fix, larvae were transferred to buffer + 200 mM glucose (pH 7-0) and the Anlagearea dissected out. From third instar larvae isolated abdomina were simply immersed infixative, after which the same procedure was followed. After washing, postfixation in 1 %osmium tetroxide with 0-5 M buffer and 300 mM D-glucose at 4 °C, the material was dehydratedand embedded in Araldite. Sections were cut on a Huxley microtome and stained for 15 minin a saturated solution of uranyl acetate in 50% ethanol, followed by 10 min in lead citrate.Sections were examined in a Philips 200 or 300 electron microscope and photographed onAgfa Scientia film or Ilford EM-4 plate.

DESCRIPTION

Life history

The chronology of larval development at 25 °C and relative humidity 90% is shownin Fig. 1. The following description concerns the histoblasts of the fifth abdominalsegment through the 3 larval instars, up to eversion of the head and thoracic imaginal•disks; other abdominal segments (excluding the last, eighth) were essentially the same.

Imaginal histoblasts in Calliphora larvae 311

Several larvae were studied in whole mounts at each of the following stages: o, 6 and13 h in the first instar; 17, 23 and 30 h in the second instar; and throughout the thirdinstar, with particular notice to timed stages at 38, 48, 66 and 90 h before pupariation,during which period synchrony between larvae becomes inevitably looser. Puparialstages were retimed from the onset of pupariation and intrapuparial third stagesstudied at [2, 10 and 16 h after pupariation (see Fig. 1). Disks are everted at 24 hpostpupariation.

Time from hatching, h

Oviposition

Hatching

1st moult

2nd moult

L,

Pupariation —

Apolysis .-prepupal

Eversion period .

Adult cuticlesecretion

Ad

-16

0

14

31

Time from pupariation, h

127

24

110

Eclosion 9-10 days

Fig. 1. Chronology of larval development at 25 °C and 90 % relative humiditythroughout life.

Organization of histoblast Anlagen within the larval epidermis

In each of the first seven segments of the larval abdomen there are 3 pairs of Anlagen,nests of small cells which even shortly after hatching (o h) are discernible in wholemounts of the epidermis as more tightly packed cells in the larval epidermis.

Counts of cell number in these earliest Anlagen approximate to the figures given byBautz (1971): 6, 8 and 3 cells in ventral (v), dorsal (d) and postero-dorsal (pd),

M. J. Pearson

Imaginal histoblasts in Calliphora larvae 313

Anlagen respectively ,(cf. Fig. 4). It is difficult always to distinguish histoblast fromneighbouring larval nuclei. Fig. 5 shows the relation of these Anlagen to the insertionsof dorso-ventral segmental muscles on either side, an important marker in locating•early stage histoblasts in either whole mount or in section.

Histoblast Anlagen become more clearly distinct during larval life as the surroundinglarval cells hypertrophy, and their nuclei become increasingly polytene. Feulgenabsorption measurements (Pearson, 1974) show that histoblasts remain diploidthroughout larval life, apart from the 4C period of cell cycle, with a nuclear diameterin the 2 C condition (G± phase) of about 6 Jim.

First instar. At 6 h there are clearly 6 histoblast nuclei at least in the ventral Anlageof the fifth segment (Fig. 2); although lying in the same plane as larval nuclei, there isalready some superposition of imaginal nuclear images due to pseudostratification,which makes accurate cell counts difficult.

At 13 h, at the end of the first instar, a granular larval cell with dense nucleus, andits smaller satellite, are visibly associated with the ventral (v) Anlage. In Fig. 3, 6-8nuclei are visible in the v Anlage, about 8 in the dorsal (d), and 4 in the smaller{pd) Anlage.

Second instar. Mitotic activity begins at or toward the end of the first instar. Thefirst moult is at 14 h.

At 17 h, the number of nuclei in each Anlage varies. Fig. 5 shows fifth segmentAnlagen with 11 © histoblasts, 13 d histoblasts, and 5 pd histoblasts, suggesting thatmost histoblasts from the previous instar have undergone division.

Cell numbers in all 3 Anlagen were found to increase throughout the second instar.Fig. 6 shows mitotic figures in a d Anlage at 23 h.

At 30 h, by the end of the second instar there are up to 24 histoblasts in the dAnlage; about 16 in the v Anlage spread around the periphery of a hypertrophic cell"which shows, in optical microscopy, a granular cytoplasm, together with a smallersatellite cell. The function of this ' granular' cell and satellite, always associated withthe v Anlage and never with d or pd Anlage, is unknown.

Fig. 2. A fifth segment v Anlage at 6 h with 7 cells grouped closely round a central cell.By comparison with later stages these represent 6 histoblasts + a satellite around the'granular' cell (cf. Fig. 3c).Fig. 2. A, pd Anlage, B, d Anlage, and c, v Anlage from the left fifth hemisegment of13-h (late first instar) larva. There are 4 histoblasts in the pd Anlage; about 8 (in-cluding a separated cell arrowed) in the d Anlage, although pseudostratification con-fuses the image; and 6 or 7 in the v Anlage.Fig. 4. The same hemisegment as Fig. 3 showing the relation of Anlagen (arrowed) tothe dorso-ventral muscle insertions.Fig. 5. A, pd, B, d, and c, v Anlagen (fifth segment) of 17-h second instar larva. Cellcounts are roughly: pd, 5 histoblasts; d, 15 histoblasts; v, about 10 cells round the' granular' cell (gr) + satellite (1). Most, if not all, histoblasts have passed once throughmitosis.Fig. 6. 23 h: a d Anlage showing histoblasts (arrowed) in mitosis.Fig. 7. 48 h: mitotic histoblasts (arrowed) in the d Anlage. Division is not synchronousthroughout the Anlage.

M. J. Pearson

Imaginal histoblasts in Calliphora larvae 315

In whole mounts of second instar larvae where the subepidermal plexus is intact,there is seen no innervation of the dorsal Anlagen; but sensory dendrites (Osborne,1963, 1964) from a local multipolar neuron terminate beneath the basement membraneat the base of ventral Anlage histoblasts (Figs. 9 c, 24).

Third instar. All 3 Anlagen are prominent in whole mounts throughout the thirdinstar. With greater numbers of cells, mitotic figures are more frequently seen(Fig. 7). However, during the late wandering phase of third instar life, mitoticactivity appears to be reduced. At 90 h (Fig. 9) there are, at an estimate, about250 cells in the ventral, 350 in the dorsal and about 100 cells in the posterodorsalAnlagen. The 2 larger Anlagen are deep islands encroaching outward among the larvalcells and over 100/im across, comprising apparently pseudo-stratified histoblasts.(Paraffin sections at this and later (intrapuparial) third instar suggest that all thethe histoblasts retain both apical and basal contact with, respectively, the cuticle andthe basement membrane.)

In the ventral Anlage, the 'granular' cell becomes peripherally displaced, and itsnucleus, which appears to undergo no further replication in the third instar, remainsdensely staining till the end of larval life. It apparently has no contact apically withthe cuticle, and does not display the granular cytoplasm seen in earlier stages (Fig. 5).

Intrapuparial third instar. After pupariation there is a rise in mitotic activity. TheAnlagen increase and encroach upon neighbouring larval cells which, occasionally,are cytolysed and rejected into the haemocoel. Neither shape nor area of the Anlagenare constant; there are at pupariation over 500 and 350-400 cells in dorsal and ventralAnlagen, respectively.

By 20 h after pupariation (in the prepupal period) the numbers of histoblasts indorsal and ventral Anlagen have increased to about 1000 and 500 histoblasts respect-ively. In sections mitotic figures are frequent (Fig. 10) and (as in the larval period)cells entering division withdraw from the basement membrane and round up whilst

Fig. 8. A, pd Anlage, B, d Anlage, and C, v Anlage of fifth hemisegment in early thirdinstar. 1 /*g colchicine in 1 fil water was injected at 38 h and the epidermis fixed 4 hlater to show mitotic activity at this stage. Colchicine-condensed metaphases are seenin all 3 Anlagen.Fig. 9. A, pd Anlage, B, d Anlage, and c, v Anlage at 90 h. All 3 Anlagen at the end oflarval growth are compact islands containing highly pseudostratified cells. In (c) is seenthe relation of sensory neurons (sn) to the v Anlage. Dendrites (arrowed) innervatethe base of the Anlage as shown in Fig. 1. t, tracheole.Fig. 10. Wax sections of a d Anlage at 24 h after pupariation (the time of the 'pupal'moult and disk eversion). A, histoblasts in mitosis arrowed. Dividing cells withdrawnfrom the basement membrane and round up whilst keeping apical cuticular contact.Division is in the plane of the epidermis, B, numerous chromatic droplets result fromdegeneration of histoblasts during rapid division. (Bouin's/haemalum and eosin;sections cut at 8 /wn.)Fig. 11. Epidermal whole mount at 17 h (second instar) showing larval epidermal cellsand campaniform sense organ with cupola insertion of the sclopale process (sc),4 nuclei and sensory neuron (sn). The same nuclei (1—4) are seen in thin section inFig. 1. These sensilla have been described in the blowfly Phormia by Osborne (1963).

316 M. J. Pearson

still maintaining their cuticular attachment. Spindles are oriented in the plane of theepidermis.

At this stage Feulgen-positive chromatic droplets appear among the rapidly dividinghistoblasts (Fig. 10). By 24 h after pupariation there are estimated to be about2500 cells in the dorsal Anlage, up to 1000 in the ventral and about 300 in the smallposterodorsal Anlage. By the time of eversion of disks there is still no migration ofhistoblasts out into the abdominal epidermis; the abdominal 'pupal' cuticle is largelysecreted by larval epidermal cells.

Mitotic activity during larval life

Fig. 12 plots the log of average cell numbers, for a number of larvae, in dorsal andventral Anlagen through larval life. In late third instars, nuclear stratification makesprecise counts in whole mount impossible; estimates are from both whole mounts and

40 •

2L 3-0h -f'a -'

? 22 0

1 0

I I 1 1 1 1 rJ U —20 40 60 80 100 120 I 140

Time, h 0 20

Fig. 12. Graph showing log of average cell numbers in dorso-lateral ( x ) andventro-lateral (O) Anlagen of a number of larvae throughout life.

8-/im sections, and in the puparial period estimates are based on 8-/im wax sectionsalone. There is a fairly constant mitotic index from the first divisions at the end of thefirst instar up to the wandering phase (mid third instar), when it declines. There isa second higher rate of increase which begins around the time of pupariation.

The collection of mitotic figures by colchicine. A method which clearly demonstratesthe number of cells entering mitosis during a given period is to arrest division atmetaphase, and to examine' in whole mount after 8 h.

One microgramme of colchicine in i [i\ water was injected into, .the third instar

Imaginal histoblasts in Calliphora larvae 317

abdomen (an average dosage of 17 /ig/g live weight, or roughly io~2 M). From 2 h upto about 10 h, typical colchicine arrests are visible, but subsequently metaphase platesfail to appear; instead, condensed chromosomes either lie scattered in the cell orclump in a chromatic mass. The number of arrests collected in an 8-h period thereforeindicates the number of cells entering M phase during the 6 h previous to collection(see Table 1). The results confirm that the mitotic index declines between 60 and90 h; that is, during the wandering phase of the late third instar.

The humoral control of histoblast activity at the end of larval life

A number of wandering third instar larvae were ligatured in the anterior abdominalregion to isolate the fifth abdominal segment from Weissman's Ring. If this operationis performed before the critical period of ecdysone release, subsequently only the

Table 1.

Age atinjection,

Colchicine arrests

No.

during the third instar

of M cells (av.)No. of cells in

h Dorsal

Anlage (av.)

Ventral

324 06 0

9 01 0 0

7/20

18/4426/c. 8042/c. 45016/c. 500

6/1712/3320/c. 5019/c. 200

—

One microgramme of colchicine in 1 fi\ distilled water was injected into larvae which werethen cultured for 8 h before preparation of whole mounts. Typical colchicine arrests appear1-5 h-2 h after injection. The number of arrests collected after 8 h therefore indicates thenumber of cells entering M during the 6 h previous to fixation. The results show averagefigures for 3 animals at each stage through the third instar (see Fig. 1).

cuticle anterior to the ligature tans to form a puparium; the posterior larval segmentsremain flaccid and do not contract into the puparial form. When such ligatured,untanned abdomina were fixed for whole mounts 4 days after pupariation of theanterior hypodermis, the histoblast Anlagen appeared much as in preparations of90-h (wandering phase) larvae. There was no great proliferation within the Anlagen;nor had histoblasts become invasive. It appears therefore that the changes of organiz-ation and behaviour within the histoblast Anlagen preparatory to metamorphosis,seen during the puparial larval period in normal animals, depend upon the hormonalstimuli which lead to puparial tanning and the pupal moult (Wigglesworth, 1970).

The histological organization and fine structure of abdominal histoblasts

In the third instar, optical and electron microscopy of sectioned material clearlyshows histoblasts engaged in larval cuticle secretion within the epidermis (Pearson,1972). The following description of histoblasts through all 3 larval instars shows thatthroughout larval life abdominal histoblasts are situated within the epidermis, andfunction as epidermal cells.

21 CHL 28

15

Imaginal histoblasts in Calliphora larvae 319

First instar. Whole mounts show histoblasts in the same focal plane as otherepidermal cells (Figs. 2-4). To find these Anlagen in section, it is important to identifythe dorso-ventral muscle insertions. Figs. 15 and 16 show a transverse section throughthe abdomen of a 6-h first instar larva, where a ventral Anlage is seen adjacent toa ventral muscle insertion. Microvillae with dense apical caps (cf. Fig. 22) are seenat the apical surface of histoblasts as well as larval epidermal cells.

Epidermal sense organs in the first instar. The campaniform organs of Calliphoralarvae are found in a mid segmental ring in each abdominal segment (Pearson, 1974).They are clearly visible in whole mount preparations (Fig. 11). They are also evidentin sections of first instar larvae. Fig. 18.shows a section through 4 nuclei of sucha sensillum (the identification of these nuclei is discussed in the legend), while Fig. 17shows both the campaniform sensory ending and the group of cells of thesensillum. Fig. 17 A, B are taken from a sequence of serial sections to demonstrate thatthe cell bodies are in fact those of a sensillum. Sensilla were found in sections of firstand second instars around the circumference, but not in association with the insertionof the segmental musculature.

Second instar. Sections of mid second instar larvae, and of late first instar larvae atthe time of laying down the early second instar cuticle (i.e. after apolys'is, beforeecdysis) show the same organization of histoblasts within the larval epidermis; inevery way comparable to first instar and third instar organization. Fig. 1 shows histo-blasts, adjacent to a muscle insertion, during the secretion of the second endocuticularlamella of the second instar cuticle.

Third instar. The histological organization of third instar histoblasts within the

Fig. 13. Thin section through a v Anlage of 12-h first instar larva, showing relation tomuscle insertion, i, insertion at cuticle; m, muscle cell; ist, first instar postapolysiscuticle; 2nd, second instar cuticle, of which already epicuticle and one endocuticularlamella are formed (see Fig. 1). h, histoblasts; /, larval epidermal nuclei.Fig. 14. The same v Anlage as in Fig. 13. This section shows histoblast (h) withapical cuticular surface (arrow).Fig. 15. A section through a v Anlage of a 6-h first instar larva at low magnification.The knife mark passes through the 'granular' cell, around which histoblasts aregrouped (cf. Figs. 2, 3 c, 5 c). h, histoblasts.Fig. 16. Apical cuticular surface of a histoblast taken from Fig. 15. The endocuticlehas separated from the microvillar (mi>) surface of the histoblast.Fig. 17. A, Araldite thick section through a cuticular sensillum (se) in a first instar larvalabdomen. B, taken from the same series of thick sections, the cell bodies of the senseorgan are shown at a distance of about 4 cell diameters from a muscle insertion. Thestructure of this sense organ is shown in Figs. 18 and 19.Fig. 18. The cell bodies (in thin section) associated with a cuticular sensillum(Fig. 17 A). Apically in this group is the body of the sensory cell (ry); Closer to theepidermal cells are the nuclei of accessory cell (ac) and neurilemma cell {nc). Thesection shows the sensory dendrite (de), surrounded by neurilemma, passing into thebody of the cupologen cell to its scolopale process. Ic, larval epidermal cell.Fig. 19. At higher magnification the sensory process is shown wrapped by neurilemma(«/)• cc, cupologen cell; nf, neuronlaments. The 4 nuclei of this'sensory group of cellsare shown in whole mount in Fig. 11.

21-2

jum24

\

21

^-M f

5 //m

H v n

23m mt

ap

Imaginal histoblasts in Calliphora larvae 321

larval epidermis, with basal attachment to the basement membrane and apical attach-ment to the cuticle, has been briefly described (Pearson, 1972). Fig. 21 shows a numberof these histoblasts in a dorsal Anlage of a 66-h third instar larva.

The Anlage comprises columnar cells with spherical nuclei about 6 /tm in diameter,the most peripheral showing epithelial continuity with the adjacent hypertrophiedlarval cells. Epithelial polarity is evident in apical and basal surface specialization, andin the intercellular junctional complexes (as in other epithelial systems: Farquhar &Palade, 1963) which are the same between histoblasts, between larval cells, and betweenhistoblast and larval cell.

Intercellular junctions. The intercellular junctional complex comprises, in apico-basal sequence, a zonula adhaerens, a longer region of septate desmosomes, occasionaldesmosomes (maculae adhaerentes), and in the basal region where the intercellularspace widens with basement membrane penetrating between cellular interdigitations,hcmidcsmosomes. Maculae occludentes (close junctions) are seen between histoblastsbelow the septate region; by analogy with other insect epidermal junctional systems(Hagopian, 1970; Poodry & Schneiderman, 1970) these are probably 2-nm gaps ratherthan tight junctions, but resolution is not good enough to be certain. Cytoplasmicbridges are occasionally seen (Fig. 23). These may be merely remnant of immediatelyprevious cell divisions, and not of further significance in the intercellular organizationof the Anlage.

Cytoplasmic fine structure: histoblasts. Cytoplasmic organization appears identicalamong all the histoblasts of the dorsal Anlage, and is typically shown in electron-micrographic section in Fig. 21. At this stage the Golgi is fairly inconspicuous,apparently engaged in the production of small vesicles (Fig. 26). The exploded con-dition of mitochondria, with the outer membrane blown out, is a condition often

Fig. 20. Araldite thick section through the d Anlage of a 66-h third instar larva.Histoblast (h) nuclei are seen within the epidermis. /, larval epidermal nuclei are seenwithin the epidermis. /, larval epidermal nucleus; m, dorso-ventral muscle insertion.Fig. 21. A montage of thin section micrographs from a d Anlage showing apical(cuticular) and basal membrane (bm) surfaces of histoblasts. In the cytoplasm short,rough cisternae are abundant. Inconspicuous Golgi (g) are seen at the base of 2 nucleiwith associated small vesicles. The microvillar (mv) surface is shown in higher mag-nification in Fig. 22. 66-h third instar. d, desmosome; n, nucleus; sj, septate junctionalregion; z, apical zonula.Fig. 22. The apical surface of a d Anlage histoblast showing microvillae. Microtubularbundles (vit) are seen in microvillae at whose tips cuticular filaments pass from dense'caps' into the youngest endocuticular lamella. (There is no evident 'subcuticle').A vesicle (ve) is apparently extruding its contents into an extracellular crypt. Similarlysized vesicles with dense contents are seen throughout the cytoplasm. At 66 h.Fig. 23. A cytoplasmic bridge between histoblasts seen in oblique longitudinal section.ap, apical histoblast surface; mt, microtubule bundle. At 66 h.Fig. 24. The basement membrane (bm) and basal surface of a v Anlage histoblastshowing termination of sensory dendrites (d): d1 is surrounded by a Schwann cell(SIP) and has penetrated the basement membrane; the naked terminal end of dendrited% lies in a groove in the base of a histoblast. hd, hemidesmosome; n, histoblastnucleus. At 66 h.

34*

25

M. J. Pearson

26 mim

Imaginal histoblasts in Calliphora larvae 323

encountered in insect epidermal preparations, e.g. Locke (1967), and probably anartifact of fixation. A few isolation membranes are seen (Locke, 1966) (Fig. 25).Microtubules are loosely oriented in apical-basal direction throughout the cytoplasm.

The apical border of the histoblasts (Fig. 22) is extended in numerous microvilli,at the tips of which cuticular fibrils arise from dense caps. Vesicles often seen openingin the extracellular crypts are similar to those produced by the Golgi system.

The endocuticle secreted by the histoblasts shows the typical Bouligand lamellateprofile (Fig. 21) (Bouligand, 1965). These lamellae are continuous above histoblastsand larval cells. There is no change in pattern or dimensions. The number of laminaeand their orientation is therefore identical throughout the cuticle, indicating thatformation of endocuticle at the surface of both polytene larval cells and histoblasts isunder identical control.

A fine-structural comparison with larval epidermal cells. In sections of mid thirdinstar epidermis, the enormous nuclei of larval cells, showing banded polytenestructure, contrast with the diploid spherical histoblast nuclei. The structure ofcytoplasm, however, is very similar. At their apical surface, larval epidermal cells showexactly the same functional organization as histoblasts; dense microvillae engaged atthe tip in the organization of endocuticular lamellae.

The intrapuparial third instar: cytological reorganization of histoblasts. At the end of

Fig. 25. Isolation membranes (im) are occasionally seen in third instar histoblasts. Inthis 66-h d histoblast an isolation membrane surrounds an electron-lucent globule.Fig. 26. Cytoplasmic profile of a 66-h d histoblast basal to the nucleus (n) showingribo8omes, rough ER elements and Golgi (g) with vesicles. Compare Fig. 27.Fig. 27. Supranuclear region of a histoblast 20 h after pupariation. Rough ER profilesshow longer profiles after intrapuparial reorganization; in this section such profilesare not fully developed (cf. Fig. 28). Both apically and basally these ER cisternae arevery frequently seen associated with vesicular Golgi (g), and at this stage with thenuclear envelope (arrow), n, nucleus.Fig. 28. An oblique section through a d Anlage at 20 h after pupariation. Two endo-cuticular lamellae of the 'pupal' cuticle (c) are already formed. ER is relativelysparser than in prepuparial third instar except in the supra- and subnuclear vicinitywhere long profiles are concentrated, frequently in association with Golgi. Hugesecondary lysosomes are common (ly). Nucleoli (nu) are large and dense.Fig. 29. A more oblique section at the margin of a 20-h d Anlage. Secondary lysosomes(ly) are abundant and enormous (see also Fig. 30). The distribution of ER at either poleof the nucleus is not seen in this more transverse section, la indicates characteristicstacking of parallel intracellular membranes which precedes cellular degeneration ofadjacent larval epidermal cells. Preparation for larval cell death is evident at this stagebefore histoblasts migrate, g, numerous vesicular Golgi concentrations; It, lipidgranules.Fig. 30. Secondary lysosomes from Fig. 29 at higher magnification, together witha degenerated cell to which other histoblasts attach by numerous cytoplasmicprocesses. In this degenerate cell dense granular cytoplasmic material is seen in theleft section, and heterogeneous components in the right section (a lobe of the samecell); the highly condensed nuclear chromatin is not in section. The secondarylysosome (/yi) is apparently at a later stage of its development than /y,. In ly, can beseen rough reticular membranes and mitochondria; in />>i are dense bodies whichmay derive from mitochondria, other granular material, and membranous whorls.

324 M. J. Pearson

the third instar, the tanned larval cuticle becomes separated from the epidermis exceptfor an inner layer of endocuticle (subsequently separated as the 'ecdysial membrane').During this period, and during the period of secretion of the 'pupal' cuticle, thehistoblasts undergo intense activity within the Anlagen; not only of cell division, butat the intracellular level there is considerable reorganization.

By the late third instar, the increase in cell numbers within the Anlagen leads toa stratified appearance of nuclei in section. In paraffin sections of intrapuparial stagesit appears that many, and possibly all histoblasts retain both apical contact with thecuticle, and, except during division, basal contact with the basement membrane(Fig. 11). This is not seen, of course, in slightly oblique thin sections cut for electronmicroscopy (Figs. 28, 29).

At 20 h after pupariation, rough endoplasmic reticulum is still present apically withscattered short cisternal profiles; and the apical surface is still folded in microvillararrangement for cuticle secretion. (This is the 'pupal' cuticle.) At the perinuclear andbasal levels of the Anlage the ER has been extensively reorganized. Rough ER is nowsparse except for aggregates of longer, convoluted profiles concentrated at theimmediate apical and basal ends of the nuclei (Fig. 27). These aggregates are usuallyassociated with a Golgi apparatus which is much more prominent than in mid thirdinstar. In many cells too there is seen close association and occasional continuity withthe outer nuclear membrane (Fig. 27).

Lipid granules are seen at this stage, and apically, large membrane-bound spaces(whose contents may have been removed in preparation).

Secondary lysosomes are numerous and large, containing a range of membranousand dense granular elements, suggesting that autophagic processes are involved in thereorganization of histoblasts. Whole cells can also degenerate to produce chromaticdroplets (Pearson, in preparation).

In the nucleoplasm, enormous nucleoli show the same structure of inner finelypacked fibrillar core surrounded by looser granules of about 20 nm diameter whichhas been described in the spheroid nucleoli of amphibian oocytes (Lane, 1967). Thereis evidently great synthesis of ribosomal precursor material in histoblast nuclei atthis stage.

DISCUSSION

The histological organization of abdominal histoblast Anlagen

There is an obvious discrepancy between the account of histological organizationof histoblasts during the first instar given here and that given by Bautz (1974). Thisdiscrepancy raises the question: who is looking at what?

In this study, the guiding criterion for the identification of histoblast Anlagen hasbeen their juxtaposition to dorso-ventral muscle insertions in transverse sectionsthrough the segment. This feature is clearly established by study of whole mountpreparations. Sections of first instar larvae (where Anlagen are difficult to locate) maybe compared in this regard to those of third instar larvae (Figs. 15, 20). The firstinstar Anlagen thus identified show in profile a group of closely packed cells, columnar

Imaginal histoblasts in Calliphora larvae 325

in comparison with the slightly hypertrophied larval cells surrounding them, at leastsome of whose apical surfaces are cuticular. This description in section - which isagain seen at the end of the first instar when second instar cuticle is being secretedafter apolysis (Fig. 14) - corroborates the whole mount image of Anlagen where thehistoblast nuclei lie in the same focal plane as larval nuclei, unlike the nuclei ofcuticular sense organs which lie in a different focal plane (Fig. 11).

The almost identical appearance of Fig. 18 to Bautz' (1974) fig. 2 suggests that theyare sections of the same structures. In this study, the serial tracing of this structure(a) backward into a nerve axon and (b) forward toward a campaniform sensillum(Fig. 17) shows that this is without doubt a cuticular sense organ. It seems likely,therefore, that Bautz has misidentified the imaginal Anlagen in first instar epidermis.The histological organization through first and second instars does not differ fromthat of the third instar, which has been described previously (Pearson, 1972). Thehistoblasts are already functional epidermal cells after hatching.

In addition to the similarity of histological organization, the fine structure ofhistoblasts does not differ greatly through the 3 larval instars until the puparial periodof activity preparatory to epidermal invasion. Nor do profiles of thin sections differgreatly from those of neighbouring larval epidermal cells; loose cisternal profiles,Golgi and vesicles (and particularly those vesicles seen opening to the cuticular surface)all indicate that both kinds of epidermal cell are identically engaged in the synthesisof cuticle throughout the early part of larval life. This activity has been described insome detail by electron-micrographic autoradiography (B. Gupta, M. Pearson &N. Cooper, unpublished).

Histoblast activity at the end of larval life

At pupariation, among a whole range of physiological changes induced by hormonalsecretion (Wigglesworth, 1970) the histoblasts begin to divide rapidly within theAnlagen. The change in mitotic activity at this stage is clearly shown by the log plot ofcell number increase (Fig. 2), following the relative quiessence of the late thirdwandering phase. Throughout this period of rapid division histoblasts apparentlymaintain their apical surface contact. At the cytological level, this mitotic activity isconcomitant with marked reorganization: a lysosomal activity during which ER isapparently removed into secondary lysosomes, and ER of a different appearance isdeveloped at apical and basal poles of the nuclei.

It seems likely that this reorganization — either directly or indirectly - is a result ofendocrine activity of Weissman's ring. The mitotic activity of the Anlagen is shown byligature to be dependent on hormonal influence from these glands (and also theappearance of chromatic droplets). It is possible that the appearance of chromaticdroplets reflects the same change of organization of healthy histoblasts at this stage,when secondary lysosomes and residual bodies become prominent. The relationbetween chromatic droplets and intracellular autophagic activity will be discussedelsewhere (Pearson, in preparation).

326 M. J. Pearson

The mitotic activity of Calliphora histoblasts through larval life: a comparison withDrosophila

The genetic analysis of adult abdominal pattern determination in Drosophila(Garcia-Bellido & Merriam, 1971; Guerra, Postlethwait & Schneiderman, 1973;Lawrence et al. 1977) has been complemented recently by a histological study ofhistoblasts through larval life (Madhiavan & Schneiderman, 1977). Clonal analysis ofthe larvae shows there is no growth of the histoblasts of dorsal (Garcia-Bellido &Merriam, 1971; Guerra et al. 1973) or ventral (Lawrence et al. 1977) Anlagen untilthe time of pupariation. There are 12-13 cells in the ventral, and about 20 histoblastcells in the two dorsal groups from hatching until pupariation (Madhavan &Schneiderman, 1977).

The condition in larval Calliphora is clearly different. Two distinct phases of mitoticactivity can be identified: (a) a steady increase in cell number from the end of the firstinstar to the wandering (mid third instar) stage, which tails off before (b) a more rapidincrease in cell number, associated with cytological reorganization and the productionof chromatic droplets shortly before the 'pupal' moult and subsequent epidermalinvasion. This second mitotic phase corresponds to the initiation of histoblastdivisions in Drosophila at or shortly before pupariation.

Bautz (1971) described the mitotic activity of Calliphora histoblasts during larvalgrowth, but considered that division was restricted, before the third instar, to themoult period, with one round per moult. The appearance of mitotic figures through-out the second instar (Fig. 6) is incompatible with this suggestion. Cell divisions arenot synchronized between histoblasts of one Anlage after the first division, norbetween Anlagen of the same or consecutive segments. (There are obvious differences•when cell counts are compared between Anlagen in several larval whole mounts atany stage.) There is seen no critical period of mitotic increase in early third instar(Bautz, 1971) although, with increasing cell numbers, mitotic figures are of course morefrequently seen. The average cell cycle time during larval growth, up to the wanderingstage, is about 15 h (Fig. 12).

Counts of colchicine arrests show a decrease of mitotic index during the wanderingphase (Table 1); a decrease which is clearly reflected in Fig. 12. Data here refer to thefifth segment, whereas Bautz studied first and second segments; nevertheless, firstsegment Anlagen do not differ significantly from the fifth either in whole mount orsection. Cell number estimates for late third instar Anlagen are based on total countsof nuclei seen in all 8-/tm sections through the Anlage; and since some of these nucleiwill appear in both of consecutive sections the result is unlikely to be an underestimate.

After pupariation, mitosis proceeds more rapidly, with an average generation timeof 8-9 h (Fig. 2).

These observations lead to the conclusion, then, that while abdominal histoblastsin Calliphora show the same organization as in Drosophila (Madhavan & Schneiderman,1977), within the larval epidermis throughout larval life, they differ in showing a phaseof larval mitotic activity. At hatching, the number of histoblasts in Calliphora appearsto be half that reported in Drosophila: 8 + 3 in the dorsal Anlagen against about 20 in

Imaginal histoblasts in Calliphora larvae 2>21

Drosophila; and in the ventral Anlage 6 against 12 in Drosophila. By the late thirdinstar, however, Calliphora has about 350 + 100 histoblasts in the dorsal and about250 in the ventral Anlagen, before the onset of mitotic activity at the time ofpupariation.

I am grateful to Dr Brij Gupta for help and for the use of electron-microscope facilities in theDepartment of Zoology, Cambridge. The work was supported at various junctures by an SRCResearch Studentship; the Department of Zoology, Cambridge, and Denis Weaver (to whomI am particularly grateful); and an SRC Fellowship held at the University of Sussex. I thankDr Peter Lawrence for criticizing the manuscript.

REFERENCES

BAUTZ, A.-M. (1971). Chronologic de la mise en place de l'hypoderme imaginal de l'abdomende Calliphora erythrocephala Meigen. (Insecte, Diptere, Brachycere.) Arclis Zool. exp. gen.112, 157-178.

BAUTZ, A.-M. (1974). Aspect ultrastructural des ilots d'histoblastes t6gumentaires de l'abdomende Calliphora erythrocephala au cours du premier stade larvaire. C. r. hebd. Sianc. Acad. Set.,Paris 279, 811-813.

BOULIGAND, Y. (1965). Sur une architecture forsaddc r£pandue dans les nombreuses cuticulesd'arthropodes. C. r. hebd. Seanc. Acad. Set., Paris 261, 3665-3668.

DOBKNDORFER, A. (1971). Untersuchungen zum Anlageplan und Determinationszuztand derweiblichen Genital- und Analprimordien von Musca domestica L. Willielm Roux Arch.Entw Mech. Org. 168, 142-168.

FARQUHAR, M. G. & PALADE, G. E. (1963). Junctional complexes in vaiious epithelia. J. CellBiol. 17, 375-412-

GARCIA-BELLIDO, A. & MERRIAM, J. R. (1971). Clonal parameters of tergite development inDrosophila. Devi Biol. 26, 264-276.

GUERRA, M., POSTLETHWAIT, J. H. & SCHNEIDERMAN, H. A. (1973). The development of theimaginal abdomen of Drosophila melanogaster. Devi Biol. 32, 361-372.

HAGOPIAN, M. (1970). Intercellular attachments of cockroach nymph epidermal cells.J. Ultrastruct. Res. 33, 233-244.

LANE, N. J. (1967). Spheroidal and ringnucleoli in amphibian oocytes. J. CellBiol. 35,421-434.LAWRENCE, P. A., GREEN, S. M. & JOHNSTON, P. (1977). Compartmentalization and growth of

the Drosophila abdomen. (In the Press.)LOCKE, M. (1966). Isolation membranes in insect cells at metamorphosis. J . Cell Biol. 31, 132A.LOCKE, M. (1967). What every epidermal cell knowns. In Insects and Physiology (ed. J. W. L.

Beament & J. E. Treherne), pp. 69-82. Edinburgh and London: Oliver and Boyd.MADHAVAN, M. & SCHNEIDERMAN, H. A. (1977). Histological analysis of the dynamics of growth

of imaginal discs and histoblast nests during larval development of Drosophila melanogaster.(In the Press.)

OSBORNE, M. P. (1963). The sensory neurones and sensilla in the abdomen and thorax of theblowfly larva. Q. Jl microsc. Sci. 104, 227—241.

OSBORNE, M. P. (1964). Sensory nerve terminations in the epidermis of the blowfly larva.Nature, Lond. 201, 526.

PEARSON, M. J. (1972). Imaginal disks and the abdominal histoblasts oiCalliphora erytJirocepliala(Diptera). Nature, Lond. 238, 349-351.

PEARSON, M. J. (1974). The abdominal epidermis of Calliphora erytltrocephala (Diptera).I. Polyteny and growth in larval cells. J. Cell Sci. 16, 113-131.

PEARSON, M. J. (1977). Pattern and polarity of sclerites in adult abdominal segments ofCalliphora erythrocephala (Diptera): anlage rotation experiments. J. Embryol. exp. Morph.35, 91-104.

POODRY, C. A. & SCHNEIDERMAN, H. A. (1970). The ultrastructure of the developing leg ofDrosophila melanogaster. Wilhelm Roux Arch. EnttoMech. Org. 166, 1-44.

URSPRUNG, H. & NOTHIGER, R. (eds). (1972). The Biology of Imaginal Discs. Berlin, Heidelberg,New York: Springer-Verlag.

WIGGLESWORTH, V. B. (1970). Insect Hormones. Edinburgh: Oliver and Boyd.

(Received 23 May 1977)