Mekkara Mandaravally Madhavan and Kornath Madhavan 1 Introduction

The epidermal cells, a derivative of ectoderm during embryogenesis of insects, contribute to the distinct cuticular pattern and form of the different stages that appear during their ontogeny. The type of cuticular products is the result of gene expression of the individual epidermal cells that lie immediately underneath these outgrowths (1). In hemimetabolous insects, the larval epidermal cells (LECs) present at the time of hatching from the egg and their descendants are responsible for the different cuticular patterns seen in the nymph and adult. In contrast, in many holometabolous insects, such as Drosophila, the distinct and different cuticular patterns exhibited by the larva and adult have a dual orgin; that of the larva is derived from the LEC and that of the adult is derived from the imaginal discs (2). The prospective integument of the adult head, thorax, genitalia, and analia is derived from imaginal discs (3-5). Each of the adult abdominal segments is derived mainly from three pairs of diploid histoblast nests (i.e., a pair of anterior dorsal [ADN], posterior dorsal [PDN] and ventral [VN] nests), located among the polytene LEC of the abdominal segments of the larva (see Figs. 1, 2A-C, and 3A,B). In addition, there is a pair of inconspicuous spiracular nests (SN) in each of the abdominal segments; during metamorphosis these nests (see Fig. 3C) develop into the paired spiracles on the lateral sides of the adult abdominal segment (3,6-9).

In the following, we describe the contributions of histoblasts in the formation of adult abdominal segments of Drosophila. The tergal area of each of the abdominal segments, depending on the presence or absence and the type of cuticular outgrowths, show distinct regions (see Fig. 4A,B). Histological studies and deletion of different histoblast nests indicate (8,10,11) that the descen-dents of the ADN form the hairy and bristled region of the tergum, whereas

From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ

VN ADN PDN SC

Fig. 1. Camera lucida drawing of a whole mount of the epidermis of the fourth abdominal segment of a 44 h old larva (same segment as shown in Fig. 2A). The whole mount was prepared after dissecting the larva mid-ventrally, treated with Feulgen's reagent, which tints the nuclei and shows the distribution of the paired anterior dorsal (ADN), posterior dorsal (PDN), and ventral (VN) histoblast nests among the larval epidermal cells (LECs). HO, ventral bands of hooks; SC, cluster of small cells of unknown function; PT, polytene cell in the midst of the VN. (From ref. 7, reproduced with permission of Springer-Verlag.)

VN ADN PDN SC

Fig. 1. Camera lucida drawing of a whole mount of the epidermis of the fourth abdominal segment of a 44 h old larva (same segment as shown in Fig. 2A). The whole mount was prepared after dissecting the larva mid-ventrally, treated with Feulgen's reagent, which tints the nuclei and shows the distribution of the paired anterior dorsal (ADN), posterior dorsal (PDN), and ventral (VN) histoblast nests among the larval epidermal cells (LECs). HO, ventral bands of hooks; SC, cluster of small cells of unknown function; PT, polytene cell in the midst of the VN. (From ref. 7, reproduced with permission of Springer-Verlag.)

those of the PDN contribute to the intersegmental membrane and the acrotergite (12) respectively (see Fig. 4B). The paired VN give rise to the sternum of the abdominal segments. The sternum contains a median sclerotized patch of cuticle, the sternite, with bristles and hairs, whereas the remaining area, the pleura, contains only hairs. The spiracles derived from the spiracular nests are located contralaterally in the pleura (see Fig. 5A). The location of these nests underneath the larval cuticle can be recognized externally in the third instar larvae and early puparia, by their closeness to larval muscle attachment sites, which appear as small depressions on the cuticle (see Fig. 5B).

The microscopic hairs, which decorate the tergite, sternite, and pleural regions, show distinct morphological features. The tergite and sternite hairs are long and thin with a narrow base and their shafts appear to arise sharply from the general body cuticle (see Fig. 6A,B). In contrast, the cuticle around the bases of the pleural hairs is membranous and thrown into folds. As a result, these hairs appear broad based (see Fig. 6E). The shaft of the pleural hairs appears forked with unequal arms (see Fig. 6D).

One of the main questions in the development of organisms is to understand, in cellular and molecular terms, how the component cells cooperate to generate a specific size and pattern in the resulting tissue or organ, and we are beginning to understand this. The development of the integument of the abdomen of Drosophila is suited for such studies because the histoblasts are an integral part of the larval abdominal epidermis and appear as a single layered epithe-

Drosophila Dorsal Organ

Fig. 2. (A) Whole mount of the right fourth abdominal hemisegment of a 44 h old larva showing the anterior dorsal (ADN), posterior dorsal (PDN), and ventral (VN) nests of histoblasts (enclosed by the dashed line), ventral band of hooks (HO), and cluster of small cells (SC) of unknown function. (B) Whole mount of the abdominal epidermis of the second segment of a 44-h-old larva showing the left anterior dorsal histoblast nest (ADN, enclosed by dashed line) among the larval epidermal cells (LECs). (C) Tangential section passing through the epidermis of the third abdominal segment of a 17-h-old larva showing the ventral nest (VN, enclosed by dashed line). See the polytene cell (PT) in the midst of the histoblasts, and the surrounding LECs. All preparations were stained with Feulgen's reagent and counterstained with fast green. (From ref. 7, reproduced with permission of Springer-Verlag.)

Fig. 2. (A) Whole mount of the right fourth abdominal hemisegment of a 44 h old larva showing the anterior dorsal (ADN), posterior dorsal (PDN), and ventral (VN) nests of histoblasts (enclosed by the dashed line), ventral band of hooks (HO), and cluster of small cells (SC) of unknown function. (B) Whole mount of the abdominal epidermis of the second segment of a 44-h-old larva showing the left anterior dorsal histoblast nest (ADN, enclosed by dashed line) among the larval epidermal cells (LECs). (C) Tangential section passing through the epidermis of the third abdominal segment of a 17-h-old larva showing the ventral nest (VN, enclosed by dashed line). See the polytene cell (PT) in the midst of the histoblasts, and the surrounding LECs. All preparations were stained with Feulgen's reagent and counterstained with fast green. (From ref. 7, reproduced with permission of Springer-Verlag.)

lium and thereby provides an excellent opportunity to examine cell-cell interactions and cell communication in a flat epithelial sheet (see Fig. 1). Further, this epithelium is more amenable to surgical manipulations and whole-mount

Fig. 3. (A) Appearance of the anterior dorsal (ADN), posterior dorsal (PDN), (B) ventral (VN), and (C) spiracular nests (SN) of wild type (18 h after pupariation) in whole-mount preparations stained with Feulgen's reagent. (From ref. 9, reproduced with permission of Springer-Verlag.)

histological preparations, compared to the pseudostratified epithelium of imaginal discs. Because there is no muliplication of the LECs or the histoblasts during the entire larval life of Drosophila, the spatial pattern established during late embryonic development is maintained in the larval epidermis. Thus, it is possible to uncouple mitosis from other processes occurring during the 96-h-long larval life. During metamorphosis, the histoblasts divide and begin to replace sequentially the LECs that undergo programmed cell death (apoptosis) (8,10). This facilitates visualization of the sequence of interactions, over a long period, of these two cell types side by side as they occur.

We now review the limited number of published studies on histoblasts, which illustrate their suitability for probing many problems in developmental biology. We also indicate where lacunae exist in the nonutilization of this model system for such studies.

When the different histoblast nests are deleted by y-irradiation, the surrounding LECs survive metamorphosis and secrete cuticle and cuticular outgrowths

Fig. 4. (A) Scanning electron microscopic (SEM) picture of the dorsal view of a stretched abdomen of a female adult fly exposing the fairly wide and usually folded intersegmental hairless regions, alternating with tergites, which are decorated with cuticular outgrowths. (B) An enlarged view of the posterior of the third and anterior of the fourth tergite and the intersegmental region showing the details of the cuticular landscape. AHR, anterior hairy region, AT, acrotergite, ISM, intersegmental membrane, MA, macrochaeta, MI, microchaeta, PHR, posterior hairy region. (From ref. 8, reproduced with permission of the Company of Biologists.)

Fig. 4. (A) Scanning electron microscopic (SEM) picture of the dorsal view of a stretched abdomen of a female adult fly exposing the fairly wide and usually folded intersegmental hairless regions, alternating with tergites, which are decorated with cuticular outgrowths. (B) An enlarged view of the posterior of the third and anterior of the fourth tergite and the intersegmental region showing the details of the cuticular landscape. AHR, anterior hairy region, AT, acrotergite, ISM, intersegmental membrane, MA, macrochaeta, MI, microchaeta, PHR, posterior hairy region. (From ref. 8, reproduced with permission of the Company of Biologists.)

(hairs) characteristic of their positions in the abdominal segment (see Fig. 7A-D). This indicates that the LEC may contain the blueprint for the adult abdominal cuticlar pattern (13). Whether this information is transmitted to the histoblasts and, if so, how that is done are details yet to be worked out.

Formation of the tergite and median sternite by the paired histoblast nests also offers an opportunity to analyze the roles of mitosis, cell growth, cell migration, and cell death in histoblasts in the realization of the final size of these sclerotized cuticular tissues. So far, no studies have been published on these aspects.

During metamorphosis of Drosophila, one of the intrinsic signals that allows the replacement of LECs by the histoblasts could come from differential titers of juvenile hormones and ecdysones. Although there is no record of studies on

Sternite Drosophila

Fig. 5. (A) Whole mount of the unstained abdominal cuticle of an adult female fly. The abdomen was cut mid-dorsally and the cuticle was spread to show the median sternites (ST), pleura (PL), and paired spiracles (S). T, cut portion of tergite. (B) Diagrammatic representation of the relative positions of the three histoblast nests and nearby muscle attachment sites in the left side of a hemisegment of a third instar larval epidermis. (From ref. 10, reproduced with permission of Springer-Verlag.)

Fig. 5. (A) Whole mount of the unstained abdominal cuticle of an adult female fly. The abdomen was cut mid-dorsally and the cuticle was spread to show the median sternites (ST), pleura (PL), and paired spiracles (S). T, cut portion of tergite. (B) Diagrammatic representation of the relative positions of the three histoblast nests and nearby muscle attachment sites in the left side of a hemisegment of a third instar larval epidermis. (From ref. 10, reproduced with permission of Springer-Verlag.)

the role of ecdysones on the dynamics of growth and differentiation of histoblasts, a few studies indicate that juvenile hormone and its synthetic analogs do affect mitosis in them and secretion of adult abdominal cuticle and its outgrowths (14-17).

Mitotic recombination using X-ray irradiation during different stages of embryonic and postembryonic development has been used extensively to gen-

Fig. 6. SEM pictures of adult abdominal hairs showing their distinct morphology: (A) tergal hairs and (B) sternal hairs. These are long and narrow and project abruptly from the cuticle. (C) The relative positions of the spiracle in the pleura and the tergo-pleural border line (arrow). PL, pleural region; S, spiracle; T, tergal region. (D) Note that the cuticle around the bases of the pleural hairs is thrown into folds and the hairs appear broad based, in constrast to those of the tergite and sternite. The shafts of the hairs are forked. (From ref. 8, reproduced with permission of the Company of Biologists.)

Fig. 6. SEM pictures of adult abdominal hairs showing their distinct morphology: (A) tergal hairs and (B) sternal hairs. These are long and narrow and project abruptly from the cuticle. (C) The relative positions of the spiracle in the pleura and the tergo-pleural border line (arrow). PL, pleural region; S, spiracle; T, tergal region. (D) Note that the cuticle around the bases of the pleural hairs is thrown into folds and the hairs appear broad based, in constrast to those of the tergite and sternite. The shafts of the hairs are forked. (From ref. 8, reproduced with permission of the Company of Biologists.)

Fig. 7. (A) and (B) are Feulgen-stained whole-mount preparations and (C) and (D) are SEM images of regions of abdominal segments of adults resulting from larvae that received y-radiation. (A) Posterior of the third and anterior of the fourth hemitergite and their intersegmental region. Persisting LECs, which are immediately posterior to the macrochaetae, secrete hairs, whereas those in the intersegmental region secrete smooth cuticle. (B) Pleural region. Note that the hairs secreted by the surviving polytene LECs show bulbous bases. (C) The tergopleural border (dashed line) of the left fourth hemisegment showing two different kinds of hair secreted by the tergal (t) and pleural (p) LECs. S, remnant of spiracle. (D) Third sternite region showing surviving LECs bearing hairs, which, similar to those on the tergite, are narrow, long, and without folds at their bases. (From ref. 13, reproduced with permission of the Company of Biologists.)

Fig. 7. (A) and (B) are Feulgen-stained whole-mount preparations and (C) and (D) are SEM images of regions of abdominal segments of adults resulting from larvae that received y-radiation. (A) Posterior of the third and anterior of the fourth hemitergite and their intersegmental region. Persisting LECs, which are immediately posterior to the macrochaetae, secrete hairs, whereas those in the intersegmental region secrete smooth cuticle. (B) Pleural region. Note that the hairs secreted by the surviving polytene LECs show bulbous bases. (C) The tergopleural border (dashed line) of the left fourth hemisegment showing two different kinds of hair secreted by the tergal (t) and pleural (p) LECs. S, remnant of spiracle. (D) Third sternite region showing surviving LECs bearing hairs, which, similar to those on the tergite, are narrow, long, and without folds at their bases. (From ref. 13, reproduced with permission of the Company of Biologists.)

erate genetically marked twin spots to estimate the primordial cell numbers in, and growth dynamics of, different histoblast nests (18). Madhavan and Schneiderman (7) have also studied these features of the histoblasts from his-tological observations and have compared the advantages and disadvantages of these two protocols. More recently, FLP/FRT (see Chapter 17) and SMART (see Chapter 22) methods have been employed to generate mitotic recombination in the imaginal cells.

Although extensive studies have been done in the identification of genes, their products and their role in pattern formation, regulation of size, and cell death in the cuticular structures resulting from the imaginal discs of Drosophila, such studies have only just begun in the histoblasts. Madhavan and Madhavan (9) observed that mutation in epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase (RTK), affects mitosis, spreading and differentiation of adult epidermal cells derived from the various histoblast and spiracular nests (see Figs. 8A-C and 9). Further, the need for EGFR becomes critical after pupation, and the requirement continues throughout pharate adult development for the correct development of the abdominal integument and spiracle. It is reported that Wingless (Wg) determines tergite and sternite cell fates (19,20), and EGFR acts synergistically with Wg (20). Kopp et al. (20) have also shown that Decapentaplegic (DPP) opposes Wg and EGFR signaling, thus promoting pleural fate in the adult abdominal epidermal cells. This explains the wild-type abdominal cuticular pattern observed in the DPP mutant adult flies.

The expression of the selector gene engrailed (en) determines the posterior compartment of the tergite (21). Under the influence of en, all cells in the posterior compartment secrete Hedgehog (Hh). This protein enters into the anterior compartment of this segment and that of the following, forms concentration gradients, and at least partly dictates the stereotypic anterior-posterior landscape and affinities of cells of the adult tergite (22-25). The details of what finally controls the polarity of the cuticular outgrowths on the tergum and sternum are still unclear (26).

It is possible that the reluctance to apply histological and molecular histo-logical methods to this system could be the result of the difficulty in making planar whole-mount preparations of histoblasts and LECs, and in the identification of the smaller and fewer cells of histoblast nests during larval stages (see Fig. 1) and metamorphosis. We believe that our stepwise description of the methods on whole-mount preparation of the integument during different stages of development of Drosophila will enable the reader to obtain a flat preparation, wherein the location of different histoblast nests and the surrounding LECs can clearly be seen after conventional nuclear and cytoplasmic staining, or specific molecular and immunological staining protocols can be applied for diverse analyses of these two types of cell, during the epigenesis of the abdominal segments.

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