Cellular Differentiation in a Highly Specialized Insect Secretory Organ ELIZABETH JOHNSON and SPENCER J. BERRY Biology Department, Wesleyan University, Middletown, Connecticut 06457, USA

Received July 1976lRevised January I977

The colleterial glands of insects are accessory reproductive structures which produce secretions that are applied to eggs after fertilization and which serve a number of protective functions. The colleterial glands of lepidoptera are of particular interest in the study of the events of cellular differentiation because they undergo rapid development, generally during the pupal adult transformation, and con- tain highly specialized cells which produce large amounts of a restricted variety of secretory pro- ducts. The extreme specialization of these organs facilitates parallel studies of differentiation at the biochemical and morphological level. This communication describes the changes in the ultrastructure of cells which will form the protein-secreting segment of the colleterial gland of the moth Actius l u m during the period of transition from the undifferentiated state to the acquisition of secretory ability.

An initial stage of general cellular proliferation by mitosis in the presumptive colleterial tissue mass is followed by differentiation of the cells in the absence of further mitosis. Four distinctive cell types are recognized during the phase of differentiation. These types include a chitogenous cell which forms the chitin lining of the main duct, and three cells which cooperate in the formation of a secretory apparatus. Cell A forms two temporary flagella-like structures which assist in the formation of a ductule, which eventually leads from the secretory cell to the main duct. Near the end of the differentiative phase, Cell A degenerates and is phagocitized by Cell B. Cell B becomes the actual secretory element, and acquires cytoplasmic features such as extensive rough endoplasmic reticulum and Golgi apparatus which are associated with synthesis and secretion of protein. The final element, Cell C, remains associated with the ductule which it helps to construct and which traverses its cytoplasm.

Introduction

Spectacular discoveries in molecular genetics have pro- vided the tools with which to probe problems of the control of cellular differentiation. One of the major problems facing students of differentiation is a lack of detailed knowledge about specific events in the morpho- genesis of specialized organs which are suitable- for bio- chemical and macromolecular analysis. We have exam- ined the ultrastructure [ l ] and the secretory activity [2, 31 of the differentiated cells of a specialized cellular organ, the colleterial gland of silkmoths. Colleterial glands are found in a wide variety of insects, and their function is to secrete various types of coatings for the newly laid eggs 141. In cockroaches, these coatings make up the complex ootheca [51, while in silkmoths the secre-

tion is applied directly to the eggs and causes them to adhere to the substrate on which they are laid. In both these cases, the product of the colleterial gland consists of protein which is cross-linked by quinone in the pres- ence of atmospheric oxygen.

The protein-secreting segment of the silkmoth colle- terial gland is of more than routine interest because a modest number of cell types is represented, and at least one of these, the secretory cell, is highly specialized mor- phologically and functionally. The entire sequence of differentiative events in colleterial tissue occurs during the transition from pupa to adult, and thus accurate de- termination of the stage of development of the gland is facilitated by inspection of the external features of the

Differentiation 8, 39-52 (1977) - 0 by Springer-Verlag 1977

40 E. Johnson and S . J. Berry:

pupa. At least one major biochemical event in the se- quence of differentiative steps can be examined in detail, because the major export protein consists of a single glycine-rich species. This protein can be detected by electrophoresis of colleterial cell homogenates after in- cubation with radioactive glycine [31. In this communi- cation we describe the changes in cell morphology which occur during the transformation of the colleterial anlage to the stage which immediately precedes the ini- tiation of the synthesis of the specialized secretory prod- uct.

Methods

lumen and the secretory cells per se. In the timetables cited above, day 0 refers to the first signs of retraction of the pupal leg anlagen from the overlying pupal cuticle. By day 0 which may be as many as ten days after trans- fer from the cold, extensive activation of biochemical pathways and reorganization of organelles has taken place, but these are not obvious from external inspection of the pupa. Very little cellular differentiation is detected during this period, and for the purposes of this report, day 0 will be considered as the point of departure for morphogenesis.

Day 0 of Development

Initiation of adult development was induced by transferring diapaus- ing pupae ofActias h a to 25” C after storage for several months at 4’ c. Colleterid anlagen and glands were dissected from more than 40 female pupae at various stages of the pupal-adult transformation. The approximate stage of development was determined by compar- ing external and internal morphological features with those referred to in timetables for the development of H. cecropia [6] and A. PO@-

When the first signs of leg retraction are distinguishable, the anlage of the colleterial gland consist of a single layer of undifferentiated epithelial cells ( ~ i ~ . 1 b). The cytoplasm contains scattered mitochondria, nonmembrane-bound ribosomes and a distrib- uted mesh of microtubules. Large deposits of glycogen

phemus F71. Pupae were transected between abdomen and thorax, and colle-

terial glands were extirpated from the abdominal half while it was submerted in ice-cold Ringer solution. Glands were fixed in toto in ice-cold 4% glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for YO min. After two brief rinses in buffer, the tissues were cut into small pieces and post-fixed for 30 min in 2% osmium tetroxide, dehydrated in acetone, and embedded in Epon-Araldite using the rapid infiltra- tion technique of Hayat 181. Thick ( 3 p) sections for light microscopy were stained with Toluidine Blue. Thin sections were made on an LKB Ultrotome 111 and picked up on naked or formvar coated grids. All sections were stained with alcoholic uranyl acetate and lead ci- trate [ Y I and were examined with a Philips 300 electron micro- scope.

Results

The colleterial tissues of A . Zuna complete cellular differ- entiation during the first 15 of the 21 days between the visibly detectable end of pupal life and the emergence of the adult moth. When pupae are first removed from the cold, the anlage of the gland consists of a pair of small sacs composed of undifferentiated cells attached to the tissue which will form the common oviduct. When the adult emerges from its cocoon, the gland consists of a pair of tubes ten centimetres or more in length. The gland shown in Figure l a was dissected from an animal at day 7 and the secretory and collecting regions are clearly established. The secretory region of the mature organ is made up of three cell types: chitogenous cells which secrete the chitinous lining of the lumen, ductule cells which form a passageway from secretory cell to

and small areas of rough endoplasmic reticulum are lo- cated in the basal cytoplasm. Many large vesicles con- taining small amounts of glycogen and flocculent mate- rial are located both between adjacent plasma mem- branes and within the cytoplasm at the apical ends of the cells. Centrioles are also prominent in the apical cy- toplasm (Fig. lc).

Mitotic Phase (Days 1-4)

The five days following day 0 are characterized by mi- totic activity which results in elongation of the entire gland anlage. The elongation of post-mitotic cells results also in an increase in the width. The first mitoses are oriented with the spindle parallel to a tangent to the lumen (Fig. Id). Each dividing cell contains large gly- cogen deposits which often lie near the spindle and are distributed to each daughter cell (Fig. Id). Following division, residual bodies (Fig. 2a), occluded by bundles of microtubules, are observed near the lumen, connect- ing adjacent cells (Fig. 2b). Later, an inter-cellular band of microfilaments develops and encircles the entire lumen (Fig. 2c).

Toward the end of the proliferative phase, a 90° change in the orientation of the spindle is apparent. One daughter cell still maintains its attachment to the lumen, but the axis of the spindle is at right angles to the pre- vious axis (Fig. 2d). This second phase of mitosis would correspond to the “determinative divisions” described by Lawrence 1101.

Differentiation in an Insect Gland

Fig. la-d. (Legend see page 43)

41

Fig. 2a-d. (Legend see page 43)

Differentiation in an Insect Gland

After mitosis is complete, the cells elongate, but maintain their attachment at the lumen. The migration of nuclei away from the lumen to different levels pro- duces a pseudostratified epithelium. During the process of cell elongation, the centrioles persist and are located at the apical end (Fig. 3a).

43

cell which is temporarily unconnected to the lumen mi- grates apically and reattaches. The cytoplasm of the outer cell wraps around the more central cells and seams are formed by the fusion of the leading edges of cyto- plasm. These seams then disappear and continuous con- centric rings of cytoplasm are seen in cross-sections of the clusters (Fig. 3c). The band of microfilaments disap- pears during the rearrangement and this results in the formation of cell-contact areas consisting of zonula oc- cludens and septate desmosomes. All the cell borders which impinge upon the lumen develop short microvilli with dense caps, which appear to be responsible for the fibrous coating on the free surfaces. For purposes of clarity, the inner cell of the cluster has been designated “A”, the median cell “B”, and the outer cell “C”. Each cell type has a distinctive role in the formation of the functional gland and we will consider the subsequent career of each cell separately.

Diffeerentiative Phase (Days 5-15)

During the last stages of cell elongation and the second mitotic phase which overlap somewhat, the apical cyto- plasm becomes much less dense because of a sparser distribution of free ribosomes. Longitudinally oriented microtubules also become an increasingly prominent feature. Mitosis ceases after day 5 and all further devel- opment involves growth and rearrangement of cells and the acquisition of specialized organellar structures.

The primary rearrangement involves the formation of concentric clusters of three cells. These clusters are separated by presumptive chitogeneous cells at the bor- der of the lumen. The cluster cells are products of the differentiative divisions at the end of the mitotic phase. Clusters of two cells are produced first (Fig. 3b) and the third outer cell is added very shortly afterwards. The initial cluster may form after division, as the daughter

Fig. la-d. a Colleterial gland including collecting regions and com- mon duct from day 1 animal. The tubular region which may be up to 15 cm long in the adult, is composed of secretory cells surrounding a central lumen. The collecting regions, clearly defined at this stage, store the secretory product until eggs are deposited ( x 5) b Undiffer- entiated tissue at day 0. A single layer of short columnar cells sur- rounds the lumen at this stage. Intra and intercellular vacuoles (v) characterize the apical region. The nucleus is centrally located and the basal cytoplasm contains small deposits of glycogen (sly) ( x 6200). c Cross-section of a centriole at day 0. Centrioles are invariably located at the apical end of the cell near the plasma mem- brane (x 4800). d Dividing cell in telophase during the mitotic phase (days 1-4). Cell division always takes place at the lumen (lu) and the first phase of divisions is parallel to a tangent to the lumen. The large pool of glycogen (gly) is distributed between the daughter cells fol- lowing cytokinesis (x 9400)

Fig. 2a-d. a Residual bodies (RBI, day 1. These structures com- posed of remnants of spindle and plasma membrane, accumulate near the luminal surface of the gland following mitotic activity in this area (x 17,200). b Spindle microtubules and residual body joining daughter cells following cytokinesis (x 26,800). c Longitudinal sec- tion of apical cytoplasm at day 5. During the period of cellular growth and elongation microfibrilar (mfl bundles are observed at the apical border of the cells. The bundles are thickest near the desmo- somes (0) and appear to form a continuous plate (arrows) around the luminal surface (lu) (x 8700). d Cell in anaphase late in the mitotic phase. Spindles during this period are perpendicular to the lumen (111) (x 5800)

Cell A: Formation of Pseudoflagella. The innermost cell assists in the formation of the ductule by Cell C, and of the secretory apparatus by Cell B. The relationship of the cluster cells early in the differentiative phase is dia- grammed in Figure 4. All three cells are attached to the lumen for a short period, but Cell A draws away and retracts basally (Fig. 5a). The apical junctions between the three cells are maintained and as Cell A withdraws from the lumen, it leaves behind an open passageway within the cytoplasm of Cells B and C. The apical cyto- plasm of Cell A throughout this process is characterized by a highly organized network of longitudinally oriented microtubules (Fig. 3c). In the earliest stages of the with- drawal, a cohesive fibrous matrix fills the opening and extends for a distance into the lumen (Fig. 5b). This matrix appears to be secreted by dark-tipped microvilli on the surface of Cells B and C in particular (Fig. 6a). Whether this is a precursor of the cuticle which will later be applied to these internal surfaces is not clear.

During this period, Cell A produces a pair of struc- tures similar to those termed “pseudoflagella” by Sel- man and Kafotis [ l l] and ‘‘cilia’’ by Barbier [ 12, 131. They originate from paired apical basal bodies, derived from contrioles (Fig. 5a), which have no accessory an- choring structures, such as rootlets, but contain the standard complement of nine outer triplet fibres. The pseudoflagella appear to grow from the apical surface of Cell A. Cross-sections through the fibrous matrix show a pair of membranous sheaths which contain a variable number of microtubules (Fig. 5c).

As differentiation proceeds, the fibrous matrix is lost, the ductule narrows, and two pseudoflagella fill the narrowed ductule and extend into the lumen (Figs. 6b

Fig. 3a-c. (Legend see page 45)

Differentiation in an Insect Gland 45

this position, is only grazed. An extra peripheral singlet is present even in a section nearly adjacent to the basal body.

Cell A continues its basal movement as the pseudo- flagella elongate until the entire cell lies within the basal cytoplasm of Cell B (Fig. 8). This process leaves behind a cylindrical opening through Cells B and C described earlier, and occupied by the pair of elongated pseudofla- gella (Fig. 6c). At this point Cell A begins to degenerate and large portions of cytoplasm and finally the nucleus are phagocytized by Cell B. In an earlier article [ll these bodies in the colleterial cells of Cecropia were misinter- pred as evidence of autodigestion products of the secre- tory cell (Cell B).

Fig. 4. Relationship of cluster and chitogenous cells during early differentiation. Three cells form the cluster (A, B, C) and chitogenous cells alternate with cell clusters along the luminal surface of the gland. Cell A forms the core and is completely encircled by Cell B. Cell C , in turn, encircles the apical half of Cell B. At this stage Cell A has withdrawn from the lumen and Cells B and C have involuted to form a wide ductule which is fdled with a fibrous plug

and 7). The arrangement of microtubules in the pseudo- flagella becomes quickly disorganized along the length of the shaft. The doublets separate and the number of fibres becomes quite variable. Figure 3c shows a cross- section of one pseudoflagellum so close to the basal body that the second basal body, somewhat forward of

Fig. 3a-c. a Apical tips of several cells at day 5. Cells are roughly hexagonal in cross-section and microfibrils (arrows) are most promi- nent at the periphery of the cell and near desmosomes. Single and paired centrioles lie just basal to the microfibrilar plate ( x 18,400). b Slightly oblique section through a two-cell cluster during the early differentiation. The luminal surface of the main cell (Cell A ) is cov- ered with short microvilli and the cytoplasm contains cIosely packed longitudinal microtubules. Cell B, which encircles Cell A, has fewer microtubules and a few microvilli bordering on the lumen. Zonulata occludens (ZO) join the two cells. The centriole (c) is visible in Cell B (x 13,300). c Cross-section through a three-cell cluster. One pseudo- flagellum @$ is visible, the other lies below the plane of this section. The 9 + 2 microtubular arrangement is evident [an extra peripheral microtubule is also visible (arrow)]. The cytoplasm of Cells A and B is rich with microtubules as compared with Cell C. One of the Cell B centrioles (c) is present sectioned at an oblique angle. Note septate desmosome (SO) (x 26,700). Membrane fusion of the leading edge of Cells B and C is complete and no seam is evident

Cell C: Ductule Formation. The outermost cell, Cell C, remains attached to Cell B at the apical junction as the other two cells migrate toward the basal border. As the matrix surrounding the pseudoflagella is lost and the ductule narrows, short microvilli covering the plasma membrane bordering on the ductule secrete a sheet of cuticle as the lining of the duct (Fig. 6c). This lining is resistent to digestion by 10% KOH and thus is pre- sumed to consist of chitin. In A . Zuna this cell persists in the adult and continues to surround the ductule. The size of cell and nucleus decreases as the gland matures and in the adult this cell is distinguishable from the chi- togenous cell only by its location.

Cell B: Secretory Apparatus Formation. As Cell A re- tracts through the cytoplasm of Cell B, the secretory apparatus develops in the apical region of Cell B. Short microvilli protrude from the plasma membrane into the opening created by the withdrawal of Cell A and encir- cle the two pseudoflagella (Fig. 9a). In apical regions of the secretory apparatus these microvilli secrete fibrous material which forms the ampulla (Fig. 6c) which is continuous with the chitinous lining of the ductule. More basally, however, the microvilli continue to elongate and form a pallisade around remnants of the pseudoflagella (Fig. 9b). The mature secretory apparatus is centrally located in the cell and anchored in position by bundles of microtubules which attach it to the cell membrane (Fig. 9c). The pseudoflagella degenerate during forma- tion of the secretory apparatus as Cell A is being di- gested and phagocytized in the basal regions of Cell B (Fig. 10a). Two hollow cylinders, left behind following pseudoflagellar degeneration, persist until the onset of secretory activity (Fig. 10a). Cell B cytoplasm, rich in ribosomes and mitochondria, develops an extensive complex of rough ER and numerous Golgi bodies prior

Fig. 5a-c. (Legend see page 48)

Fig. 6a-c. (Legend see page 48)

48 E. Johnson and S. J. Berry:

Fig. 7. Apical portion of cell cluster after pseudoflagella formation. The ductule formed by Cells B and C has narrowed and two pseudo- flagella, which originate at the paired basal bodies in Cell A , extend down the narrow duct and into the lumen

Fig. 5a-c. a Apical portion of cell cluster and adjacent chitogenous cell (Cc) before formation of the pseudoflagella. Microfibrilar (mf) bundles are found in chitogenous cell cytoplasm but are no longer seen in the cluster cells. Longitudinal microtubules predominate in Cells A and B and microvilli are developing along the luminal surface of all cells. Apical centrioles are present in all three cluster cells but in Cell A, a second pair are in the process of formation (x 14,200). b Fibrous matrix urn) formed as Cell A retracts from the luminal surface. The cytoplasm of Cells B and C forms a cylinder around this plug and microvilli on the plasma membrane of these cells elabo- rate the fibrous material and secrete the dense droplets associated with it. Junctional complexes bind the apical regions of all four cell types (x 14,000). c Fibrous plug in cross-section. Two membranous sheaths (ms) occupy the center of the plug. One is empty and the other contains three microtubules (x 19,000)

Fig. 6s-c. a Microvilli on the surface of Cell C. Fibres are visible in space between tips of the microvillae and the cuticle lining of the forming duct (x 21,600). b Pseudoflagella (pfl originating at the basal body and extending through the ductule formed by Cells B and C. The ductule has narrowed and fibrous material @m) still sur- rounds the pseudoflagella (x 22,000). c Ductule from a day 10 ani- mal. Cell C surrounds the ductule and cuticular lining is secreted by dark tipped microvilli. The chitin lining is continuous with that pro- duced by the chitogenous cell (Cc) at lower right and left. At the basal end of the ductule lies the cbitogenous ampulla (um) being secreted by short microvilli in Cell B. Pseudoflagella (pfl still occupy the centre of the ductule and degenerative changes are evident partic- ularly towards the lumen (x 11,200)

Fig. 8. Cell cluster following basal withdrawal of Cell A. The elon- gating pseudoflagella, formed as Cell A withdraws basally, occupy the center of the ductule formed by Cell C. The secretory apparatus will be formed in apical regions of Cell B as Cell A retracts com- pletely and begins to degenerate. Portions of degenerating Cell A cytoplasm (AA) are located in the basal cytoplasm

Fig. 9a-c. a Short microvilli (mv) protruding from the plasma mem- brane of Cell B in the early stages of secretory apparatus formation. The microvilli surround the two pseudoflagella (pfl which temporar- ily fill the opening created by the withdrawal of Cell A. Each pseudo- flagella is surrounded by a dense matrix (x 27,000). b Slightly ob- lique section through a nearly mature secretory organule of a day 11 animal . The pseudoflagella have degenerated, endoplasmic reticu- lum is widespread throughout Cell 13 cytoplasm (x 12,300). c Apical cytoplasm of Cell B and secretory apparatus. Microtubular bundles (arrows) run to the plasma membrane and anchor the mature secre- tory apparatus in position (x 9600)

Fig. IOa-c. a Basal cytoplasm of Cell B in a day 12 animal contain- ing extensive endoplasmic reticulum and remnants of Cell A . Nuclei, at the bottom of the micrograph, are centrally located in the cell and contain several large nucleoli (Nu) ( x 8300). b Cross-section through a mature secretory apparatus. Two hollow cylinders occupy the centre of the apparatus following dcgeneration of the two pseudofla- gella ( x 15,600). c Cytoplasm of Cell B during active secretion. Golgi bodies (Gb) and associated vesicles (v) are prominent. Devel- opment of endoplasmic reticulum reaches a peak after active secre- tion has commenced (x 20,500)

Fig. 9a-c. (Legend see page 48)

Fig. IOa-c. (Legend see page 48)

Differentiation in an Insect Gland 51

of the wax-moth Galleria mellonella [12, 131. In these cases the central cell of the cluster produces flagellar structures which assist in the formation of a cuticle-lined duct [ I l , 13, 171. Although these examples are limited to secretory structures in lepidoptera, Sreng and Quen- nedey [ 181 have described a similar sequence of events in the differentiation of cockroach tergal glands. The final configuration is typical of secretory organs and or- ganules in a great variety of insect orders. A few exam- ples of secretory structure of similar organization in- clude the cockroach colleterial gland [ 191, defensive glands of beetles [201, hypodermal glands of bugs [211, pheromone glands of scorpion flies [22], There is no information on the morphogenesis of these structures, but the organization of the secretory apparatus and ducts suggests a similar developmental history. The con- centric arrangement of cells is also reminiscent of the tormogen-trichogen relationship in bristle formation and the socket-scale precursor relationship in scale forma- tion.

The orientation of the plane of mitosis is consistent with the observations of Lawrence 101 that proliferative divisions occur with the spindle in a plane parallel to the prospective cuticular surface, while differentiative divi- sions occur in a plane at right angles to this surface. Thus Cells A and B, which serve the most highly spe- cialized functions, are formed by differentiative divisions and initially have no contact with the lumen. It is the migration of the cytoplasm of these cells to the lumen which results in the formation of the characteristic con- centric clusters.

The function of the band of microfilaments at the border of the lumen during the elongation stage may be to stabilize the tubular configuration before the deposi- tion of the cuticular lining. Wessells et al. [231 have im- plicated microfilaments in the formation of the oviducal glands of chickens, and others [24, 251 noted similar bands which are thought to be responsible for the for- mation of the neck of bottle-shaped cells during embryo- genesis. In the colleterial gland, the tubular configura- tion is established before the microfibrillar bands ap- pear, but the outward growth during cell elongation may produce stresses which would cause the cells to detach and form a truly stratified epithelium if the apical ends were not anchored in this manner.

Formation of pseudoflagella is apparently rather loosely organized by the basal bodies, since the classic 9 + 2 arrangement breaks down along the shaft. It seems clear that this rather flaccid structure has no mo- tile function and serves as a passive core around which the duct is organized. Selman and Kafatos [ 111 suggest that the pseudoflagella serve some organizational func-

to the onset of secretory activity on day 13 (Fig. 10c).

Chitogenous Cells. In the adult these cells line the lumen of the gland and secrete the cuticular lining on that sur- face. They first appear as distinct cells following cluster formation. They are recognizable, not only because of their location on the lumen, but because they develop extensive rough ER early in the differentiative phase (Fig. 5a, b). Microvilli develop on the lumenal surface of these cells and secrete a heavy layer of chitin. This layer, which forms the lining of the lumen, is continuous with the lining of the ductule produced simultaneously by Cell C. Following deposition of the chitin the volume of cell and nucleus decreases to one-fourth the presecretory size.

Discussion

A general pattern of cellular differentiation during orga- nogenesis is apparent from consideration of the develop- ment of the colleterial gland. This pattern is typical of the general plan of formation of hypodermal organules such as bristles and scales [14, 151, and most recently in the Galea of another silkmoth, Actius polyphemus [ 1 1 I. The initial stage is characterized by cellular proliferation by mitotic activity, during which little morphogenetic “progress” is evident. There is some overlap, however, and in the colleterial gland cells, the border of daughter cell nearest the lumen has already begun to form micro- villi while anaphase of a determinative division is being completed. A stage of growth and differentiation in the absence of further mitotic activity follows the prolifera- tive phase. During this period, the ploidy of the cells increases by endomitosis, and specialized structures are formed. The final stage of this process is, of course, the initiation of functional activity. The beginning of special- ized synthetic activity occurs at day 13 with the appear- ance of detectable amounts of secretory protein in ho- mogenates of the gland, (Grayson, unpublished data). This general pattern has been observed for other insect organs, including the silk gland of the larval silkmoth [ 161, and may reflect a general response of differentiat- ing cells to the hormonal environment.

A more specific pattern of differentiation of secre- tory organs and organules associated with cuticle is ap- parent. The formation of concentric clusters of cells dur- ing the post-mitotic period as a prelude to secretory or- ganule formation has been reviewed recently [ 181, and are referred to as “class 3” cells. A similar type of or- ganization has also been reported in the colleterial gland

52

tion in the formation of the cuticle of the duct, and noth- ing in our observations would contradict this idea. The composition of the fine fibrous network of the plug asso- ciated with the pseudoflagella is unknown, but it resem- bles the fibrous cuticle precursor associated with the mi- crovillar borders. The material of the plug seems partly gelled or polymerized because the plug retains its char- acteristic protuberant shape during the processing of the tissue. As Cell A begins withdrawing toward the basal border, the plug seems to disperse, and is lost altogeth- er.

Although the disappearance of the pseudoflagella is noted by Selman and Kafatos [ill and Barbier [131, actual destruction of the cell was not observed. In fact, Selman and Kafatos find that the cell bearing the pseu- doflagella is transformed into one of two zymogen cells. The inner cell (A) of the silkmoth colleterial seems to have a more restricted developmental potential and de- stroys itself after serving as a mold for duct formation. Thus the synthetic activities of each of the cells demon- strates the expression of a different genetic program which is presumably activated by the same hormonal stimulus exerted early in the last instar.

E. Johnson and S . J. Berry

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