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Cuticle differentiation during Drosophila embryogenesis
Arthropod Structure & Development 35 (2006) 137e152 www.elsevier.com/locate/asd
Cuticle differentiation during Drosophila embryogenesis Bernard Moussian*, Christof Seifarth, Ursula Mu¨ller, Ju¨rgen Berger, Heinz Schwarz Max-Planck-Institute for Developmental Biology, Department for Genetics and Microscopy Unit, Abteilung III, Spemannstrasse 35, 72076 Tu¨bingen, Germany Received 31 March 2006; accepted 11 May 2006
Abstract The constitutive criterion for the evolutionary successful clade of ecdysozoans is a protective exoskeleton. In insects the exoskeleton, the socalled cuticle consists of three functional layers, the waterproof envelope, the proteinaceous epicuticle and the chitinous procuticle that are produced as an extracellular matrix by the underlying epidermal cells. Here, we present our electron-microscopic study of cuticle differentiation during embryogenesis in the fruit fly Drosophila melanogaster. We conclude that cuticle differentiation in the Drosophila embryo occurs in three phases. In the first phase, the layers are established. Interestingly, we find that establishment of the layers occurs partially simultaneously rather than in a strict sequential manner as previously proposed. In the second phase the cuticle thickens. Finally, in the third phase, when secretion of cuticle material has ceased, the chitin laminae acquire their typical orientation, and the epicuticle of the denticles and the head skeleton darken. Our work will help to understand the phenotypes of embryos mutant for genes encoding essential cuticle factors, in turn revealing mechanisms of cuticle differentiation. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Drosophila; Embryogenesis; Epidermis; Cuticle; Apical undulae
1. Introduction Apical extracellular matrices (aECM) of epithelial cells are pivotal for the function of epithelia. In many cases, aECM have a stereotype organisation to fulfil their physiological or developmental role. The cuticle of insects is an aECM that protects the animal against environmental harm and dehydration. In addition, it serves as an exoskeleton assisting in locomotion. Cuticle differentiation starts during late embryogenesis and continues until the establishment of the imaginal cuticle involving complete renewal during larval and pupal (holometabolous insects) or nymphal stages (hemimetabolous insects) (Truman and Riddiford, 1999). The properties of the cuticle may differ between developmental stages or between different body regions. For instance, larval cuticle produced during embryogenesis is usually soft, whereas imaginal thoracic cuticle is harder. Beneath the
* Corresponding author. Tel.: þ49 7071 601 476; fax: þ49 7071 601 384. E-mail address: [email protected] (B. Moussian). 1467-8039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2006.05.003
cuticle lies a monolayer of epidermal cells that produces and secretes cuticular components respecting the demands of the different stages of development. During embryonic cuticle differentiation the epidermal cells undergo concomitant differentiation as they change their shape and develop strong cell-cell contacts to withstand tension and pressure during life outside the eggshell. Histological descriptions of cuticle differentiation have been published for many hemimetabolous as well as holometabolous insects (Locke, 1966; Guthrie and Tindall, 1968; Locke, 1969; Hillman and Lesnik, 1970; Ziese and Dorn, 2003). In summary, the cuticle consists of three layers, the outermost waterproof envelope, the middle protein-rich epicuticle and the inner chitinous procuticle that contacts the apical plasma membrane of epidermal cells through the so-called assembly zone (Locke, 2001). Parallel running chitin microfibrils that contain about 17e20 chitin fibres (Neville, 1975) are arranged in sheets, the so-called laminae, that are stacked helicoidally (Bouligand, 1965) (Fig. 1). Modifications that account for physical properties of the different types of cuticles occur in the epicuticle and the upper zone of the procuticle, often called the exocuticle.
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Fig. 1. Schematic view of chitin organisation in the insect cuticle after Bouligand (1965). Parallel chitin microfibrils (black lines) are arranged in sheets, the so-called laminae (squares). One chitin microfibril in each lamina has been highlighted in red. The laminae are arranged in helical stacks. By consequence, in oblique sections (bracket) the microfibrils appear to form apicalbasal arches.
In contrast to the histological studies on cuticle differentiation, molecular and genetic works are scarce in this field. Only recently, factors required for procuticle production and assembly have been identified in Drosophila. For instance, Retroactive (Rtv) and Knickkopf (Knk), both unknown proteins, are necessary for chitin microfibril formation (Moussian et al., 2005b, 2006). Moreover, mutations in the Drosophila chitin synthase-1 gene krotzkopf verkehrt (CS-1/ kkv) have revealed that an intact procuticle is necessary for epicuticle formation and stability (Moussian et al., 2005a). In fact, many more factors essential for Drosophila cuticle differentiation await identification and characterisation making the Drosophila embryo a suitable model system to study de novo cuticle differentiation. For a genetic and molecular analysis, a detailed description of cuticle differentiation in the wild-type embryo is required. Indeed, Hillman and Lesnik have studied Drosophila embryonic cuticle differentiation already in 1970. According to their description, Drosophila cuticle differentiation proceeds as in other insects producing the layers step by step. However, several important questions were not addressed in their work. How, for instance, are chitin microfibrils and laminae oriented in the course of procuticle assembly? Does the specialised cuticle of the denticles develop as the adjacent epidermal cuticle? To complement and extend the study of Hillman and Lesnik (1970), we investigated cuticle differentiation on physically
B. Moussian et al. / Arthropod Structure & Development 35 (2006) 137e152 Table 1 Brief summary of the stages of embryogenesis during which cuticle differentiation takes place Stage Hours 15 16a 16b 17a 17b 17c 17d
Characteristics
12 13 15 16 17
Dorsal closure, head involution, tracheal luminal chitin Chitin in the head skeleton Chitin in the epidermis Exoskeleton and pharynx visible Emergence of Malpighian tubules, start of labrum melanisation, tracheal luminal chitin vanishes 18.25 Malpighian tubules thicken, air-filled tracheae, melanisation of H-piece and mouth hooks 19-hatching Pigmentation of ventral denticles and head skeleton
fixed embryos using the modern method of cryo-immobilisation that preserves specimen integrity better than chemical fixation methods (Mu¨ller and Moor, 1984; Studer et al., 1989; McDonald and Morphew, 1993). This approach allowed us to analyse chitin lamina orientation and to compare cuticle differentiation in naked and denticle-forming epidermal cells. 2. Materials and methods 2.1. Fly embryos Flies of the wild-type stocks Samarkand, Lausanne (both for TEM) and OregonR (for SEM) and the mutant stocks kkvIB22 and Ddcn7, both carrying loss-of-function mutations, respectively, were cultured at 25 C in cages on apple-juice agar plates garnished with a spot of yeast. Embryos were staged using the timetable of Campos-Ortega and Hartenstein (1985). All wild-type stocks were obtained from the Bloomington Stock Centre at the University of Iowa/USA; the mutant stocks were obtained from the Tu¨bingen Stock Centre, Germany. 2.2. Light and fluorescence microscopy For observation of living embryos with Nomarski optics a Zeiss Axiophot II microscope equipped with a 20 objective (Plan Neofluar, NA 0.5) was used. For the analysis of cuticle differentiation and melanisation, the cuticle preparation technique was applied (Wieschaus and Nu¨sslein-Volhard, 1986). Staged Eggs were dechorionated in bleach and treated with Hoyer’s medium that clears soft tissue leaving behind the vitelline membrane and the cuticle. For observation of these embryos with Nomarski optics a Zeiss Axiophot II microscope equipped with a 20 objective (Plan Neofluar, NA 0.5) was used. For the detection of chitin by Fluostain (Sigma-Aldrich,
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Munich, Germany) dechorionated embryos were fixed for 20 min at the interface of heptane and Hepes-buffered 3.7% histology-grade formaldehyde (Sigma-Aldrich, Munich, Germany) at pH 7.0. The fixative was removed and replaced by methanol; the embryos were devitellinised by shaking, washed several times with methanol and stored at 4 C. For staining, embryos were dehydrated in phosphate buffered saline (PBS) containing 0.1% Triton X-100 (PBST). They were incubated for 12 h at 4 C in 2 mg Fluostain per millilitres PBST, washed several times with PBST and mounted in Aqua-Poly/ Mount (Polysciences). Detection of chitin was observed with a Zeiss Axiophot II microscope using fluorescence light, a 460 nm filter and a 20 objective (Plan Neofluar, NA 0.5). Pictures were taken by Zeiss Axiocam digital camera and processed with Photoshop. 2.3. Transmission and scanning electron microscopy For morphological characterisation embryos were first dechorionated in bleach and then, without removing the vitelline membrane, cryo-immobilized by high-pressure freezing. Living embryos of appropriate stages were transferred to aluminium platelets of 100 or 150 mm depth containing 1-hexadecene (Studer et al., 1989). The platelets were sandwiched with platelets without any cavity and then frozen with a high-pressure freezer (Bal-Tec HPM 010, Balzers, Liechtenstein). The frozen embryos were freed from extraneous hexadecene under liquid nitrogen and transferred to 2 ml microtubes with screw caps (Sarstedt #72.694) containing the substitution medium precooled to 90 C. Samples were kept in 2% osmium tetroxide, 0.5% uranyl acetate (prepared from a 20% stock solution in methanol) and 0.5% glutaraldehyde (prepared from anhydrous glutaraldehyde: EMS #16530, Electron Microscopy Sciences, Ft. Washington, USA) in anhydrous acetone at 90 C for 32 h, at 60 C and 40 C for 4 h at each step in a home-made freeze-substitution unit. After washing with acetone the samples were transferred into an acetone-Epon mixture at 30 C (1:1 for 4 h, 1:2 for 12 h), warmed up to room temperature, infiltrated in Epon (3 changes within 30 h) and polymerised at 60 C for 48 h. Ultrathin sections (50e70 nm) stained with 2% uranyl acetate in 70% methanol for 10 min and in 0.4% lead citrate in 0.1 N NaOH for 2 min were viewed in a Philips CM10 electron microscope at 60 kV. For WGA-labelling unstained Epon sections were incubated with biotinylated WGA (10 mg/ml, Vector Labs, Burlingame, USA) followed by rabbit anti biotin antibodies
Fig. 2. Sub-staging of wild-type embryos at the stages 16 and 17 by fluorescence microscopy (AeC) and Nomarski optics (Hoyer’s fixed animals DeI, living animals JeL). Course of chitin production and subdivision of stage 16: As detected by the chitin-binding dye Fluostain, compared to early stage 16 (16a, A), in addition to the tracheal plexus that contains chitin (tra), the head skeleton (hs) at late stage 16 (16b, B) also produces chitin. Subsequently, at early stage 17 (17a, C), the epidermis starts to produce chitin (arrow in C). Interestingly, at early stage 16, chitin can be detected in the hindgut (*). Embryos of stage 16a and 16b are both hardly visible in cuticle preparations (D,E). Subdivision of stage 17: At early stage 17 (17a, F), the pharynx emerges (arrows) and the surface of the embryo smoothens. Stage 17b is defined by the emergence of the entire head skeleton, and the epidermal cuticle clearly lines the body of the embryo (G). In the living embryo, the dorsal trunk (tra) and the Malpighian tubules (mal) become visible (J). The labrum (arrow) and the ventral denticles (triangle) start to melanise at stage 17c (H). As seen in the living animal, the dorsal trunk is now air-filled (K). Finally, at stage 17d the head skeleton is entirely melanised (I,L). (AeC,L) dorsal view and (DeK) lateral view of representative embryos. Anterior is to the left.
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Fig. 3. Ultrastructure of epidermal cells of embryos at stages 11 to 14. At stages 11 to 14 (AeD) the epidermal cells are columnar, and contain a well-developed ER network and many lipid drops. Mitochondria are often found at the apical portion of the cell. At stage 14 (D), the epidermal cells accumulate glycogen. In stages 11 (E) to 14 (F) microtubules run parallel to the apical plasma membrane, and a membranous structure called the first embryonic cuticle covers the embryos. aj, adherens junction; EC1, embryonic cuticle 1; er, endoplasmatic reticulum; gl, glycogen; ld, lipid drops; mt, microtubules; n, nucleus; vm, vitelline membrane. Scale bars 500 nm, scale bar in A applies for AeD.
(ENZO Life Sciences, Farmingdale, USA) and protein A10 nm gold conjugates (gift from Dr. York Stierhof, ZMBP Tu¨bingen). Labelled sections were then stained with 1% aqueous uranyl acetate for 3 min and lead citrate. For scanning electron microscopy embryos were fixed for five hours in 4% formaldehyde and 0.5% glutaraldehyde at 4 C. They were manually devitellinized in PBS, osmium treated (1% osmium tetroxide in 100 mM phosphate buffer, pH 7.2), dehydrated through an ethanol series, subjected to critical point drying from CO2 and sputter coated with 10 nm Au-Pd. Samples were examined at 20 kV accelerating
voltage in a Hitachi S800 field emission scanning electron microscope. 3. Results 3.1. Subdivision of stages 16 and 17 Cuticle differentiation as described by Hillman and Lesnik (1970) occurs during the commonly employed embryonic stages 15, 16 and 17 of Campos-Ortega and Hartenstein (1985). According to the latter publication, the two terminal
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Fig. 4. Ultrastructure of the differentiating cuticle at stage 15. Epidermal cells are more compact during stage 15 than in the previous stages (A). At the tips of irregular membrane protrusions fragments of the envelope are formed (B). Larger fragments of the envelope are formed at denticles (B 0 ). EC1, embryonic cuticle 1; ed, ecdysal droplets; env, envelope; gl, glycogen; mt, microtubules; n, nucleus; rif, plasma membrane riffles; vm, vitelline membrane. Scale bars in A and B 500 nm, in B 0 250 nm.
stages 16 and 17 last for about 11 h (13 to 24 h after egg laying, AEL). To have a finer staged series to standardise embryo collection for electron microscopy, we subdivided stages 16 and 17 monitoring the course of cuticle differentiation, chitin production and cuticle melanisation (tanning) on fixed and live animals by light and fluorescence microscopy. Differentiation of other morphological landmarks such as the tracheal system, the midgut and the Malpighian tubules is included to facilitate rapid staging. The data are summarised in Fig. 2 and Table 1. At stage 16 (13 to 16 h AEL, Figs. 2A,B,D,E), the embryo is completely removed by Hoyer’s medium and no cuticle is recognisable in cuticle preparations; however, chitin as a marker for the procuticle, is detectable in the head skeleton at the last third of this stage (15 h AEL). We use this criterion to subdivide stage 16 in stages 16a (13 to 15 h AEL) and 16b (15 to 16 h AEL). At the beginning of stage 17 (16 to 17 h AEL, Figs. 2C,F) the epidermal cuticle, albeit thin, and the pharynx emerge in cuticle preparations. As previously shown (Moussian et al., 2005a), chitin is detected in the epidermis and strongly in the ventral
denticles at this stage (not shown). Other striking differences between stage 16 and stage 17 embryos are the constriction of the midgut lobes and the appearance of the Malpighian tubules, both observed on living animals (not shown). Based on these differences, we call this period stage 17a. Subsequently, the dorsal trunk, albeit not yet air-filled, becomes easily discernable from the surrounding tissue (stage 17b, 17 to 18.25 h AEL, Figs. 2G,J). The head skeleton and ventral denticles remain un-melanised, but are now clearly visible. At the beginning of stage 17c, the dorsal trunk starts to fill with air, and at the end of this stage the whole tracheal plexus is air-filled (18.25 to 19 h AEL, Figs. 2H,K). Concomitantly, the ventral denticles and the labrum begin to melanise, and the Malpighian tubules darken. Finally, at stage 17d, the entire head skeleton melanises, and the animal starts to move within the egg case (19 to 22.5 h AEL, Figs. 2I,L). Embryos start to hatch 20.5 h AEL, and over three quarters of the embryos have hatched after 22.5 h AEL. To study cuticle differentiation, thin sections of cryo-immobilized embryos from stage 11 to the end of embryogenesis (including the defined sub-stages 16a and b and 17aed),
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Fig. 5. Ultrastructure of the differentiating cuticle at stage 16a. Fragments of the envelope separated by small gaps (open triangles) cover almost the entire surface of the epidermal cells that are cuboidal now (A). By contrast, the envelope is continuous at the denticles that start to form a premature electron-dense epicuticle (A 0 ), which is absent at the naked cell surfaces (B,C). As shown on cross (B) and longitudinal sections (C), the apical plasma membrane forms extended protrusions that we call apical undulae (see also Fig. 15). aj, adherens junction; bl, basal lamina; d, denticle; env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; mt, microtubules; n, nucleus; pro, procuticle; und, apical undulae; vm, vitelline membrane. Scale bars in A, B and C 500 nm, in A 0 250 nm.
were examined using a transmission electron microscope (TEM). In addition, we detected chitin by gold-labelled WGA on thin sections to follow procuticle assembly. 3.2. Cuticle differentiation from stage 11 to 14 The epidermal cells are columnar at stages 11 to 14 (Figs. 3AeD), and the nucleus lies in the middle of the cell. They are connected via adherens junctions; typical ladder-like
septate junctions, by contrast, are missing. The cytoplasm contains many ER cisternae and mitochondria that preferentially localise to the apical portion of the cell, underneath a zone of clear cytoplasm, which harbours only microtubules that are oriented in parallel to the apical plasma membrane (Figs. 3E,F). Microtubules are also abundant at the lateral plasma membrane in an apical-basal orientation. Occasionally glycogen accumulates basally; a basal lamina is not present.
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Fig. 6. Ultrastructure of the differentiating cuticle at stage 16b. The epidermal cells are cuboidal, and the fragments of the envelope have fused to a continuous layer (A). The epicuticle is now being formed also underneath the envelope of the naked cell surfaces (B). d, denticle; ed, ecdysal droplets; env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; mt, microtubules; n, nucleus; pro, procuticle; vm, vitelline membrane. Scale bars 500 nm.
Neither chitin is produced nor cuticle is formed at these stages, however instead, an irregular thin sheath covers the embryo. The space between the sheath and the epidermal surface contains only a small amount of disordered material (Figs. 3E,F). This structure was not described by Hillman and Lesnik (1970), but had been found in other dipteran embryos and may be homologous to the so-called first embryonic cuticle (EC1) observed in many insects (Truman and Riddiford, 1999).
of the riffles fragments of the outermost envelope are eventually produced. Ventrally, primordia of denticles containing cables of microtubules and probably actin filaments are emerging. Their apical plasma membrane remains smooth, however, the envelope is almost continuous at these positions (Fig. 4B 0 ). The appearance of the denticles is earlier than determined by cuticle preparations and light microscopy.
3.3. Cuticle differentiation at stage 15
Stage
Envelope
Epicuticle
Procuticlea
ECSb
Cell shape
15 16a 16b 17a 17b 17c 17d
<10 nm <10 nm <10 nm 10 nm 15 nm 20 nm 20 nm
e e 10 nm 40 nm 70 nm 120 nm 120 nm
e 25 nm 30 nm 80 nm 160 nm 300 nm 300 nm
85 nm 110 nm 130 nm 135 nm 240 nm 460 nm 460 nm
Columnar-cuboidal Cuboidal Cuboidal Cuboidal Cuboidal-flat Flat-cuboidal Flat
At stage 15, cells at the dorsal side of the embryo start to flatten (Fig. 4A), but are still columnar, whereas ventral cells remain as they were at previous stages (not shown). The nucleus moves to the basal side of the cell, where glycogen further accumulates. The cytoplasm is organised as in previous stages. The apical plasma membrane roughens irregularly (Figs. 4A,B). The apical microtubules run at the basis of these structures parallel to the cell surface. This is the stage at which cuticle differentiation starts. At the tips
Table 2 Growth of the cuticle during stages 15 to 17d
Until stage 17c, growth is continuous, therefore average thicknesses are given. a The procuticle is defined as the chitin-harbouring layer. b The extracellular space (ECS) is the widest distance between the apical plasma membrane and the surface of the envelope.
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Fig. 7. Ultrastructure of the differentiating cuticle at stage 17a. The epidermal cells are cuboidal (A). The epicuticle is now partitioned into a lower electron-dense and an upper electron-lucid sub-layer (B,C). Apical undulae are still present as observed in cross (B) and longitudinal sections (C). aj, adherens junction; env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; mt, microtubules; n, nucleus; pro, procuticle; und, undulae. Scale bars 500 nm.
No chitin is detected at this stage (Fig. 8A), and the EC1 is intact. 3.4. Cuticle differentiation at stage 16a All epidermal cells are now cuboidal (Fig. 5A). The organisation of the cytoplasm is unchanged. The nucleus is located to the basal half of the cell. The basal lamina starts to be produced. The apical plasma membrane now forms regular microtubule-containing corrugations that can be seen in longitudinal but not in cross
sections (Figs. 5B,C). Hence, both these corrugations and their microtubules run perpendicular to the anteroposterior axis of the animal. We name these structures apical undulae. They are not formed at the plasma membrane of denticle buds. For the first time, chitin is detected by gold-labelled WGA underneath the almost continuous envelope (Fig. 8B). It is, however, not possible to discern any chitin laminae (Figs. 5B,C). Obviously, detection of chitin on thin sections is more sensitive than on fixed embryos (stage 17a, Fig. 2C). There is no sign of epicuticle formation except beneath the
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envelope of denticles (Fig. 5A 0 ). The EC1 dissociates, and electron-dense droplets reminiscent of ecdysal droplets that are formed during moulting, are present apically outside the cell. In total, the EC1 covers the embryo for about five to seven hours. Interestingly, it persists during epidermal morphogenesis (dorsal closure and head involution) from stage 14 to 15 indicating its flexibility. 3.5. Cuticle differentiation at stage 16b Epidermal cells are cuboidal, and their cytoplasm remains organised as at the previous stage (Fig. 6A). The apical undulae persist (not shown), the amount of glycogen slightly diminishes, whilst procuticle formation continues (Figs. 6B and 8C, Table 2). Chitin laminae are not detectable morphologically. The electron-dense epicuticle is now seen beneath the envelope of all epidermal cells, while the electron-lucid epicuticle is not formed yet (Fig. 6B). 3.6. Cuticle differentiation at stage 17a Epidermal cells and their contents are indistinguishable from cells at stage 16b (Fig. 7A), the apical undulae are present (Figs. 7B,C). The electron-lucid epicuticle is formed between the thin electron-dense epicuticle and the envelope. Hence, at this stage, the cuticle has roughly the organisation of the mature cuticle. The procuticle thickens due to a continuous secretion of chitin (Figs. 7B,C and 8D, Table 2); chitin laminae, however, remain indiscernible. 3.7. Cuticle differentiation at stage 17b At stage 17b, epidermal cells flatten (Fig. 9A), the lateral membrane undulates, and septate junctions form ladder-like arrays. The cytoplasm is predominantly occupied by glycogen and ER cisternae. Lateral microtubules persist, but less microtubules run parallel to the surface membrane. Apical undulae are now flatter and irregularly spaced (not shown). Due to a broader epicuticle that is now clearly distinguishable from the other two layers, in addition to chitin synthesis, the cuticle thickens substantially (Figs. 9B,C and 8E, Table 2). However, chitin laminae are not visible. As observed in many other insects, chitin seems to be absent from the epicuticle. Electron-dense tube-like structures, probably precursors of the pore canals, are formed in the epi- and the procuticle (Figs. 9B,C). At late stage 17b a third electron-dense sheet is added to the envelope. 3.8. Cuticle differentiation at stage 17c Fig. 8. Detection of chitin by gold-labelled WGA (black dots in the procuticle, pro, brackets) on thin sections for TEM reveals that the formation of the procuticle starts after the onset of envelope formation at stage 15 (A) during stage 16a (B), and before the epicuticle emerges at stage 16b (C). During the stages 17a (D), 17b (E) and 17c (F) chitin amount in the procuticle increases. At stage 17d (G), chitin production seems to have ceased, as the procuticle does not grow further. Alternatively, a tighter packaging of newly produced chitin into laminae without changing procuticle thickness may mask chitin production. Scale bar in A is 500 nm and applies for AeG.
No major changes are observed in the organisation of epidermal cells at this stage compared to cells at stage 17b (Figs. 9A and 10A). The amount of glycogen decreases, and apical undulae have almost vanished (Fig. 10D). The layers grow (Table 2), chitin amount increases but laminae are still not visible (Figs. 10BeD and 8F). The lumen of the pore canals broadens (inset in 10C), and the mature envelope is
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Fig. 9. Ultrastructure of the differentiating cuticle at stage 17b. The epidermal cells are flatter than at the previous stage (A), and the lateral membranes lengthen and become wavy (arrows). Since stage 17a (Figs. 7B,C), the procuticle has thickened remarkably (B,C). The envelope maturates to a five-lamellar structure, and pore canals start to form (B,C). To visualise chitin laminae that have a helicoidal arrangement giving a parabolic texture to the procuticle, cross (B) and oblique sections (C) are compared. No chitin laminae can be seen at this stage. env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; n, nucleus; pc, pore canal; pro, procuticle. Scale bars 500 nm.
now clearly composed of five alternating electron-dense and electron-lucid sheets. 3.9. Cuticle differentiation at stage 17d At the end of stage 17d the apical plasma membrane of the flat cell (Fig. 11A) is devoid of apical undulae (not shown) and epidermal cells cease to secrete cuticle material, as ER cisternae are almost absent, suggesting that the larva is ready to hatch. The basal lamina maturates (compare to Fig. 5A). Glycogen has been entirely consumed, and the amount of chitin does not seem to change compared to the previous stage (Fig. 8G, Table 2). Chitin laminae are now fully differentiated and condensed (Figs. 11B,C). They gain their typical arrangement that gives the procuticle a parabolic texture (Bouligand, 1965) (see also Fig. 1). The arrangement of the upper half of the procuticle changes in mature ventral denticles and is rather spongy (Fig. 12A). In addition, the electron-dense epicuticle broadens
and further darkens in denticles; this probably reflects sclerotisation and pigmentation observed in cuticle preparations (Fig. 2I). However, stage 17d embryos mutant for Ddc with defects in sclerotisation and pigmentation (Wright, 1987) exhibit nevertheless darkened denticular epicuticle (Fig. 12B). Hence, sclerotisation and pigmentation are not unambiguously observable with an electron microscope. The gross morphology of denticles in chitin deficient cuticles from krotzkopf verkehrt (kkv) mutant embryos is normal (Figs. 12D,E) suggesting that chitin and the procuticle, which is almost missing in kkv mutant embryos (Moussian et al., 2005a) (Fig. 12C) are not required for shaping a denticle. 3.10. Formation and degradation of tracheal luminal chitin The tracheal luminal chitin filament has been shown to assist tracheal tube dilation during embryogenesis (Tonning et al., 2005). To verify staging of the embryos sectioned for
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Fig. 10. Ultrastructure of the differentiating cuticle at stage 17c. The epidermal cells are flat (A). The cuticle continues thickening during this stage, but no chitin laminae can be discerned in oblique (C) compared to cross sections (B). Pore canals (*) are clearly distinguishable form the surrounding cuticle (inset in C). Only shallow traces of the apical undulae are seen in longitudinal sections (D). env, envelope; epi, epicuticle; gl, glycogen; ld, lipid drops; n, nucleus; pc, pore canal; pro, procuticle; und, undulae; vm, vitelline membrane. Scale bars in AeD 500 nm, in the inset of C 250 nm.
TEM analysis we monitored the course of tracheal luminal chitin formation and degradation (Fig. 13). From stages 15 to 16b (Figs. 13AeD), the lumen of the developing trachea contains chitin fibres running parallel to the length of the tracheae. At stage 17a, electron-lucid areas emerge in-between the fibres that seem to lose their continuity (Fig. 13E). At stage 17b, only little material is left in the lumen of the tracheae (Fig. 13F). Finally, at stage 17c, the lumen of the tracheae is cleared; this is the stage when tracheae fill with air (Fig. 2K
and not shown). These observations differ slightly from data published recently by Anna Tonning and colleagues in Developmental Cell in 2005, insofar as degradation of chitin occurs later in our TEM study compared to their analysis on whole mount embryos. Interestingly, luminal chitin is removed or absorbed when the envelope entirely covers the apical surface of the cells indicating that a mechanism must exist, which allows transport of conceivably degraded chitin molecules through the differentiating tracheal cuticle.
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Fig. 11. Ultrastructure of the differentiating cuticle at stage 17d. The epidermal cell has further flattened, and does not contain any glycogen (A). The cuticle ceases thickening at this stage, when orientation of chitin laminae takes place as observed in oblique (C) sections compared to cross sections (B). Pore canals persist (inset in B). The apical plasma membrane of the epidermal cell is flat, and apical undulae are absent. bl, basal lamina; env, envelope; epi, epicuticle; hl, haemolymph; n, nucleus; pc, pore canal; pro, procuticle. Scale bars 500 nm, in the inset of B 250 nm.
4. Discussion The Drosophila larval cuticle, a three-layered aECM, is produced in the second half of embryogenesis. The events taking place during cuticle differentiation are summarised in Fig. 14 and Table 2. 4.1. Summary of cuticle differentiation 4.1.1. Embryonic cuticle 1 Prior to cuticle formation in the truest sense of the word there is an aECM that is most probably homologous to the so-called first embryonic cuticle (EC1) found in many insects (Truman and Riddiford, 1999). The function of the EC1 is still elusive. In Drosophila, it may be an evolutionary relict as in more basal insects such as Therombia domestica (silverfish) the EC1 still contains fibrous material (chitin?). In the Drosophila embryo, this structure is free from chitin.
Alternatively, as proposed by Tepass and Hartenstein (1994), it may be just the extracellular matrix that exists before cuticle differentiation. In this sense, the term ‘‘cuticle’’ may be misleading. 4.1.2. The envelope The envelope is the outermost layer of the cuticle and protects the animal from dehydration. Its precursor, consisting of two electron-dense sheets enclosing an electron-lucid one, is produced at the tips of plasma membrane riffles at stage 15, and the gaps between the fragments are closed by a yet unknown mechanism at stages 16a and 16b. No changes are observed during stages 17a and 17b, but at the transition of stage 17b to 17c, a third electron-dense sheet is formed within the electron-lucid one. The mature envelope has five alternating electron-dense and electron-lucid sheets. This is the same arrangement as observed in the envelope (‘‘cuticulin layer’’) of the Calpodes ethlius larva by Locke (1966).
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observed at stage 16b. The bulk of chitin, however, is secreted in a short time period at the stages 17b and 17c. Hence, chitin accumulation within the extracellular space seems not to be continuous, indicating a peak of chitin synthase activity at these stages. The typical parabolic texture of chitin laminae becomes visible only at stage 17d, prior to hatching. 4.2. Phases of cuticle differentiation
Fig. 12. The procuticle is not needed for denticle morphology. As seen in cross sections of the wild-type denticle, the centre of the denticle harbours the brighter extension of the procuticle that seems to be continuous with the electron-dense epicuticle, which directly contacts the envelope (A). In the Ddcn7 mutant denticle the epicuticle appears to be normal (B). The denticle of kkvIB22 mutant embryos the epicuticle is clearly separated from the remnants of the procuticle (C). The wild-type ventral denticle has a characteristic shape with the bent tip pointing to the posterior (D). The denticle of the chitin-deficient kkvIB22 mutant embryo has an identical shape (E). Note that the surface of the kkvIB22 mutant embryo is wrinkled due to the defective procuticle. Scale bars in AeC 500 nm, in D and E 1 mm.
4.1.3. The epicuticle The epicuticle emerges first at stage 16b underneath the closed precursor of the envelope. At stage 17a, it is partitioned into an upper electron-lucid and a lower electron-dense sublayer. Thereafter, it thickens continuously. At stage 17b, pore canals that yet lack a lumen are established within the epicuticle. At stage, 17c, the pore canals expand to form a lumen. 4.1.4. The procuticle Secretion of chitin to form the procuticle starts during envelope assembly at stage 16a and before epicuticle secretion can be
In summary, we propose that cuticle differentiation in the Drosophila embryo may be divided into three phases (Fig. 14). First, during the phase of establishment (stage 15 to 17a), the envelope, the procuticle (chitin) and the epicuticle are established and the layers adopt their final shape. Interestingly, the lower procuticle starts to be secreted at stage 16a before the middle epicuticle emerges at stage 16b. Next, at stages 17b and 17c, during the phase of growth the epicuticle and the procuticle substantially thicken. Finally, at stage 17d, the cuticle acquires its physical qualities as chitin laminae of the procuticle gain their typical orientation and the head skeleton and the ventral denticles melanise. The course of cuticle differentiation in the Drosophila embryo described here differs in some substantial points from that published by Hillman and Lesnik (1970). In their work, they show that cuticle differentiation is sequential, the outermost envelope being produced as the first, and the innermost procuticle as the last layer. Moreover, we show that the envelope prior to hatching is five-lamellar rather than tri-lamellar as it is in the larva of Calpodes ethlius (Locke, 1966). Our description of cuticle establishment also differs from that described for Manduca sexta embryos (Ziese and Dorn, 2003), which is very similar to that of Drosophila presented by Hillman and Lesnik (1970). We think that the differences mainly derive from the different fixation techniques affecting specimen preservation. Cryo-immobilisation followed by freezesubstitution reflects the natural state of the embryo better than direct chemical fixation followed by dehydration at
Fig. 13. Tracheal cells, as shown here for the posterior cells of the dorsal trunk, produce a luminal chitinous filament that is required for tube diameter regulation. At early stage 15 (A), luminal chitin fibres have a wavy appearance and are in close contact to the surface of the tracheal cells that form fragments of cuticular envelope (env) at late stage 15 (B). At stages 16a and b, the envelope is continuous, the chitin filament is unchanged (C,D). At stage 17a, electron-lucid patches separate the chitin fibres (E). At stage 17b, almost no chitin fibres are found in the tracheal lumen that is now almost devoid of any electron-dense material (F). env, envelope; lum, tracheal lumen; tae, taenidial fold. Scale bars 500 nm.
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room temperature. Moreover, we traced back procuticle establishment with gold-labelled WGA detecting chitin, which is more sensitive than judging upon morphological features only. Alternatively, differences in ecological strategies and life cycles of Manduca sexta (120 h of embryogenesis) and Drosophila melanogaster (22.5 h of embryogenesis) may impose different mechanisms of cuticle production.
env
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The apical plasma membrane protrudes riffles at stage 15 at the tip of which the envelope is formed. Thereafter, between stage 16a and 17b the epidermal cells form apical corrugations, the apical undulae, that are stabilised by microtubules (Fig. 15). They are arranged in an angle of 90 degree to the length of the embryo, and chitin microfibrils adjacent to the cell surface are formed perpendicular to them. As they contain microtubules they are different from the taenidial folds of the trachea (Figs. 13CeF) that lack microtubules but are fortified by actin filaments (Matusek et al., 2006). We do not find any similar cellular structure in the literature, and we suspect that they play a stabilising role before the cuticle assumes this function. In addition, they may be required for correct orientation of microfibrils that are placed in a right angle to the undulae. 4.4. Cuticle differentiation in denticles
stage 17a
Phase 2
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cross
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envelope epicuticle procuticle Fig. 14. Model of the three phases of cuticle differentiation in the Drosophila embryo. First, the three layers are established from stage 15 to stage 17a (phase 1 or phase of establishment). Microfibrils (red lines) that are produced perpendicular to the apical undulae are assembled into laminae. Second, the cuticle thickens (phase 2 or growth phase). The uniform orientation of the microfibrils within the laminae is shown in a cross section (cross) of the longitudinal view (long). Third, the chitin laminae rotate (phase 3 or phase of maturation). At the same time the ventral denticles and the head skeleton darken. In each lamina the portion of a single microfibril that lies in the plane of the drawing is highlighted in dark red. The rotation of the microfibrils is demonstrated in the cross section of the longitudinal view: in the extreme cases, a cross-sectioned microfibril in the longitudinal view appears longitudinal in the cross section and vice-versa. env, envelope; epi, epicuticle; pro, procuticle; rif, riffles; und, undulae.
The cuticle of denticles is thicker than the cuticle of naked cell surfaces, suggesting that different cellular mechanisms of cuticle differentiation may occur at these positions. Indeed, riffle and undulae formation is suppressed at those sites of the apical plasma membrane where denticles emerge, and the electrondense plaques underline the entire denticle producing plasma membrane. This suppression may enlarge the area of secretion of proteins as well as increase the density of chitin synthase machinery. Hence, riffles and undulae in epidermal cells, in contrast to the situation in gut epithelial cells, may probably not form to enlarge the secretory surface. Another speciality of the denticles is that their melanisation requires the localisation of melanisation enzymes to the denticles, prohibiting their spreading. There is no evidence how this is achieved. 4.5. Some molecular aspects of cuticle differentiation The non-sequential manner of secretion and layer-organisation implies that extracellular cuticle components to some extent possess a self-organising capacity. Namely orientation of chitin laminae seems not to occur during chitin synthesis and microfibril formation at the apical plasma membrane, but later at stage 17d when the bulk of chitin and cuticle proteins have already been deposited in the extracellular space. Similarly, self-assembling processes have been suggested to govern the formation of alternating dense and watery layers within the procuticule of butterfly pupae (Steinbrecht, 1985). Hence, in the Drosophila embryo, procuticle maturation proceeds in two steps. First, at stages 15 to 17c, chitin microfibrils are formed and arranged
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B aj
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Fig. 15. The apical undulae. Model of the apical undulae that are formed during stages 16a to 17b at the surface of epidermal cells (A). Longitudinal section of an embryo at stage 17a shows the apical undulae (und) as regular protrusions of the apical plasma membrane (B and B 0 ). Tangential section of the surface of an embryo at stage 17a reveals that chitin microfibrils run perpendicular to the apical undulae (C). Oblique-longitudinal section of an embryo at stage 17a demonstrates that long strands of microtubules (mt) underlay the apical undulae (D). aj, adherens junction; mt, microtubule; und, undulae. Scale bars in B, C and D 1 mm, in B 0 500 nm.
in parallel at the apical plasma membrane, processes that, as suggested by the mutant phenotypes (Moussian et al., 2005b, 2006), require the function of the membrane-bound proteins Knickkopf (Knk) and Retroactive (Rtv). Second, chitin laminae rotate with respect to each other at stage 17d, probably through an interaction with extracellular chitin-binding proteins. Assembly of chitin microfibrils and laminae is also influenced by the secreted chitin deacetylases Serpentine (Serp) and Vermiform (Verm) that presumably remove the acetyl-group from the chitin monomer N-acetyl-glucosamine thereby changing the physical properties of chitin (Luschnig et al., 2006). Likewise, in plants, physical as well as enzymatic reactions in the extracellular space have been proposed to be responsible for organising the network of cellulose and other polysaccharides during cell wall differentiation (Cosgrove, 2005). We are still a long way towards an understanding of the molecular mechanisms of cuticle assembly. Notably the
mechanisms of envelope and epicuticle differentiation are enigmatic. Based on the present study, the characterisation of phenotypes caused by mutations in genes coding for cuticle proteins contributes to our learning of how the insect cuticle as an example of an aECM is assembled. Acknowledgements We would like to thank Christiane Nu¨sslein-Volhard for support, and Matthew Harris for critical reading of the manuscript. References Bouligand, Y., 1965. On a twisted fibrillar arrangement common to several biologic structures. Comptes rendus he´bdomadaires des se´ances de l’Acade´mie des sciences. Se´rie D: Sciences Naturelles 261, 4864e4867.
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Campos-Ortega, J.A., Hartenstein, V., 1985. The Embryonic Development of Drosophila melanogaster. Springer Verlag, New York. Cosgrove, D.J., 2005. Growth of the plant cell wall. Nature reviews. Molecular Cell Biology 6, 850e861. Guthrie, D.M., Tindall, A.R., 1968. The Biology of the Cockroach. Edward Arnold Ltd., London. Hillman, R., Lesnik, L.H., 1970. Cuticle formation in the embyro of Drosophila melanogaster. Journal of Morphology 131, 383e396. Locke, M., 1966. The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae). Journal of Morphology 118, 461e494. Locke, M., 1969. The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid in an insect. Journal of Morphology 127, 7e39. Locke, M., 2001. The Wigglesworth Lecture: insects for studying fundamental problems in biology. Journal of Insect Physiology 47, 495e507. Luschnig, S., Batz, T., Armbruster, K., Krasnow, M.A., 2006. serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila. Current Biology 16, 186e194. Matusek, T., Djiane, A., Jankovics, F., Brunner, D., Mlodzik, M., Mihaly, J., 2006. The Drosophila formin DAAM regulates the tracheal cuticle pattern through organizing the actin cytoskeleton. Development 133, 957e966. McDonald, K., Morphew, M.K., 1993. Improved preservation of ultrastructure in difficult-to-fix organisms by high pressure freezing and freeze substitution: I. Drosophila melanogaster and Strongylocentrotus purpuratus embryos. Microscopy Research and Technique 24, 465e473. Moussian, B., Schwarz, H., Bartoszewski, S., Nu¨sslein-Volhard, C., 2005a. Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster. Journal of Morphology 264, 117e130. Moussian, B., So¨ding, J., Schwarz, H., Nu¨sslein-Volhard, C., 2005b. Retroactive, a membrane-anchored extracellular protein related to vertebrate snake neurotoxin-like proteins, is required for cuticle organization in the larva of Drosophila melanogaster. Developmental Dynamics 233, 1056e1063.
Moussian, B., Tang, E., Tonning, A., Helms, S., Schwarz, H., Nu¨ssleinVolhard, C., Uv, A.E., 2006. Drosophila Knickkopf and Retroactive are needed for epithelial tube growth and cuticle differentiation through their specific requirement for chitin filament organization. Development 133, 163e171. Mu¨ller, M., Moor, H., 1984. Cryofixation of thick specimens by high pressure freezing. In: Revel, J.-P., Barnard, T., Haggis, G.H. (Eds.), The Science of Biological Specimen Preparation for Microscopy and Microanalysis, vol. Proeceedings of the second Pfefferkorn Conference at the Sugar Loaf Mountain Resort. Scanning Electron Microscopy, Inc., Traverse City, MI. AMF O’Hare, IL 60666, pp. 131e138. Neville, A.C., 1975. Biology of the Arthropod Cuticle. Springer Verlag, Berlin Heidelberg New York. Steinbrecht, R.A., 1985. Fine structure and development of the silver and golden cuticle in butterfly pupae. Tissue and Cell 17, 745e762. Studer, D., Michel, M., Mu¨ller, M., 1989. High pressure freezing comes of age. Scanning Microscopy, Supplement 3, 253e268; discussion 268e269. Tepass, U., Hartenstein, V., 1994. The development of cellular junctions in the Drosophila embryo. Developmental Biology 161, 563e596. Tonning, A., Hemphala, J., Tang, E., Nannmark, U., Samakovlis, C., Uv, A., 2005. A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea. Developmental Cell 9, 423e 430. Truman, J.W., Riddiford, L.M., 1999. The origins of insect metamorphosis. Nature 401, 447e452. Wieschaus, E.F., Nu¨sslein-Volhard, C., 1986. Looking at embryos. In: Roberts, D.B. (Ed.), Drosophila: A Practical Approach. IRL Press, Washington, D.C., pp. 199e227. Wright, T.R., 1987. The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. Advances in Genetics 24, 127e222. Ziese, S., Dorn, A., 2003. Embryonic integument and ‘‘molts’’ in Manduca sexta (Insecta, Lepidoptera). Journal of Morphology 255, 146e161.
Cuticle differentiation during Drosophila embryogenesis Bernard Moussian*, Christof Seifarth, Ursula Mu¨ller, Ju¨rgen Berger, Heinz Schwarz Max-Planck-Institute for Developmental Biology, Department for Genetics and Microscopy Unit, Abteilung III, Spemannstrasse 35, 72076 Tu¨bingen, Germany Received 31 March 2006; accepted 11 May 2006
Abstract The constitutive criterion for the evolutionary successful clade of ecdysozoans is a protective exoskeleton. In insects the exoskeleton, the socalled cuticle consists of three functional layers, the waterproof envelope, the proteinaceous epicuticle and the chitinous procuticle that are produced as an extracellular matrix by the underlying epidermal cells. Here, we present our electron-microscopic study of cuticle differentiation during embryogenesis in the fruit fly Drosophila melanogaster. We conclude that cuticle differentiation in the Drosophila embryo occurs in three phases. In the first phase, the layers are established. Interestingly, we find that establishment of the layers occurs partially simultaneously rather than in a strict sequential manner as previously proposed. In the second phase the cuticle thickens. Finally, in the third phase, when secretion of cuticle material has ceased, the chitin laminae acquire their typical orientation, and the epicuticle of the denticles and the head skeleton darken. Our work will help to understand the phenotypes of embryos mutant for genes encoding essential cuticle factors, in turn revealing mechanisms of cuticle differentiation. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Drosophila; Embryogenesis; Epidermis; Cuticle; Apical undulae
1. Introduction Apical extracellular matrices (aECM) of epithelial cells are pivotal for the function of epithelia. In many cases, aECM have a stereotype organisation to fulfil their physiological or developmental role. The cuticle of insects is an aECM that protects the animal against environmental harm and dehydration. In addition, it serves as an exoskeleton assisting in locomotion. Cuticle differentiation starts during late embryogenesis and continues until the establishment of the imaginal cuticle involving complete renewal during larval and pupal (holometabolous insects) or nymphal stages (hemimetabolous insects) (Truman and Riddiford, 1999). The properties of the cuticle may differ between developmental stages or between different body regions. For instance, larval cuticle produced during embryogenesis is usually soft, whereas imaginal thoracic cuticle is harder. Beneath the
* Corresponding author. Tel.: þ49 7071 601 476; fax: þ49 7071 601 384. E-mail address: [email protected] (B. Moussian). 1467-8039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2006.05.003
cuticle lies a monolayer of epidermal cells that produces and secretes cuticular components respecting the demands of the different stages of development. During embryonic cuticle differentiation the epidermal cells undergo concomitant differentiation as they change their shape and develop strong cell-cell contacts to withstand tension and pressure during life outside the eggshell. Histological descriptions of cuticle differentiation have been published for many hemimetabolous as well as holometabolous insects (Locke, 1966; Guthrie and Tindall, 1968; Locke, 1969; Hillman and Lesnik, 1970; Ziese and Dorn, 2003). In summary, the cuticle consists of three layers, the outermost waterproof envelope, the middle protein-rich epicuticle and the inner chitinous procuticle that contacts the apical plasma membrane of epidermal cells through the so-called assembly zone (Locke, 2001). Parallel running chitin microfibrils that contain about 17e20 chitin fibres (Neville, 1975) are arranged in sheets, the so-called laminae, that are stacked helicoidally (Bouligand, 1965) (Fig. 1). Modifications that account for physical properties of the different types of cuticles occur in the epicuticle and the upper zone of the procuticle, often called the exocuticle.
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Fig. 1. Schematic view of chitin organisation in the insect cuticle after Bouligand (1965). Parallel chitin microfibrils (black lines) are arranged in sheets, the so-called laminae (squares). One chitin microfibril in each lamina has been highlighted in red. The laminae are arranged in helical stacks. By consequence, in oblique sections (bracket) the microfibrils appear to form apicalbasal arches.
In contrast to the histological studies on cuticle differentiation, molecular and genetic works are scarce in this field. Only recently, factors required for procuticle production and assembly have been identified in Drosophila. For instance, Retroactive (Rtv) and Knickkopf (Knk), both unknown proteins, are necessary for chitin microfibril formation (Moussian et al., 2005b, 2006). Moreover, mutations in the Drosophila chitin synthase-1 gene krotzkopf verkehrt (CS-1/ kkv) have revealed that an intact procuticle is necessary for epicuticle formation and stability (Moussian et al., 2005a). In fact, many more factors essential for Drosophila cuticle differentiation await identification and characterisation making the Drosophila embryo a suitable model system to study de novo cuticle differentiation. For a genetic and molecular analysis, a detailed description of cuticle differentiation in the wild-type embryo is required. Indeed, Hillman and Lesnik have studied Drosophila embryonic cuticle differentiation already in 1970. According to their description, Drosophila cuticle differentiation proceeds as in other insects producing the layers step by step. However, several important questions were not addressed in their work. How, for instance, are chitin microfibrils and laminae oriented in the course of procuticle assembly? Does the specialised cuticle of the denticles develop as the adjacent epidermal cuticle? To complement and extend the study of Hillman and Lesnik (1970), we investigated cuticle differentiation on physically
B. Moussian et al. / Arthropod Structure & Development 35 (2006) 137e152 Table 1 Brief summary of the stages of embryogenesis during which cuticle differentiation takes place Stage Hours 15 16a 16b 17a 17b 17c 17d
Characteristics
12 13 15 16 17
Dorsal closure, head involution, tracheal luminal chitin Chitin in the head skeleton Chitin in the epidermis Exoskeleton and pharynx visible Emergence of Malpighian tubules, start of labrum melanisation, tracheal luminal chitin vanishes 18.25 Malpighian tubules thicken, air-filled tracheae, melanisation of H-piece and mouth hooks 19-hatching Pigmentation of ventral denticles and head skeleton
fixed embryos using the modern method of cryo-immobilisation that preserves specimen integrity better than chemical fixation methods (Mu¨ller and Moor, 1984; Studer et al., 1989; McDonald and Morphew, 1993). This approach allowed us to analyse chitin lamina orientation and to compare cuticle differentiation in naked and denticle-forming epidermal cells. 2. Materials and methods 2.1. Fly embryos Flies of the wild-type stocks Samarkand, Lausanne (both for TEM) and OregonR (for SEM) and the mutant stocks kkvIB22 and Ddcn7, both carrying loss-of-function mutations, respectively, were cultured at 25 C in cages on apple-juice agar plates garnished with a spot of yeast. Embryos were staged using the timetable of Campos-Ortega and Hartenstein (1985). All wild-type stocks were obtained from the Bloomington Stock Centre at the University of Iowa/USA; the mutant stocks were obtained from the Tu¨bingen Stock Centre, Germany. 2.2. Light and fluorescence microscopy For observation of living embryos with Nomarski optics a Zeiss Axiophot II microscope equipped with a 20 objective (Plan Neofluar, NA 0.5) was used. For the analysis of cuticle differentiation and melanisation, the cuticle preparation technique was applied (Wieschaus and Nu¨sslein-Volhard, 1986). Staged Eggs were dechorionated in bleach and treated with Hoyer’s medium that clears soft tissue leaving behind the vitelline membrane and the cuticle. For observation of these embryos with Nomarski optics a Zeiss Axiophot II microscope equipped with a 20 objective (Plan Neofluar, NA 0.5) was used. For the detection of chitin by Fluostain (Sigma-Aldrich,
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Munich, Germany) dechorionated embryos were fixed for 20 min at the interface of heptane and Hepes-buffered 3.7% histology-grade formaldehyde (Sigma-Aldrich, Munich, Germany) at pH 7.0. The fixative was removed and replaced by methanol; the embryos were devitellinised by shaking, washed several times with methanol and stored at 4 C. For staining, embryos were dehydrated in phosphate buffered saline (PBS) containing 0.1% Triton X-100 (PBST). They were incubated for 12 h at 4 C in 2 mg Fluostain per millilitres PBST, washed several times with PBST and mounted in Aqua-Poly/ Mount (Polysciences). Detection of chitin was observed with a Zeiss Axiophot II microscope using fluorescence light, a 460 nm filter and a 20 objective (Plan Neofluar, NA 0.5). Pictures were taken by Zeiss Axiocam digital camera and processed with Photoshop. 2.3. Transmission and scanning electron microscopy For morphological characterisation embryos were first dechorionated in bleach and then, without removing the vitelline membrane, cryo-immobilized by high-pressure freezing. Living embryos of appropriate stages were transferred to aluminium platelets of 100 or 150 mm depth containing 1-hexadecene (Studer et al., 1989). The platelets were sandwiched with platelets without any cavity and then frozen with a high-pressure freezer (Bal-Tec HPM 010, Balzers, Liechtenstein). The frozen embryos were freed from extraneous hexadecene under liquid nitrogen and transferred to 2 ml microtubes with screw caps (Sarstedt #72.694) containing the substitution medium precooled to 90 C. Samples were kept in 2% osmium tetroxide, 0.5% uranyl acetate (prepared from a 20% stock solution in methanol) and 0.5% glutaraldehyde (prepared from anhydrous glutaraldehyde: EMS #16530, Electron Microscopy Sciences, Ft. Washington, USA) in anhydrous acetone at 90 C for 32 h, at 60 C and 40 C for 4 h at each step in a home-made freeze-substitution unit. After washing with acetone the samples were transferred into an acetone-Epon mixture at 30 C (1:1 for 4 h, 1:2 for 12 h), warmed up to room temperature, infiltrated in Epon (3 changes within 30 h) and polymerised at 60 C for 48 h. Ultrathin sections (50e70 nm) stained with 2% uranyl acetate in 70% methanol for 10 min and in 0.4% lead citrate in 0.1 N NaOH for 2 min were viewed in a Philips CM10 electron microscope at 60 kV. For WGA-labelling unstained Epon sections were incubated with biotinylated WGA (10 mg/ml, Vector Labs, Burlingame, USA) followed by rabbit anti biotin antibodies
Fig. 2. Sub-staging of wild-type embryos at the stages 16 and 17 by fluorescence microscopy (AeC) and Nomarski optics (Hoyer’s fixed animals DeI, living animals JeL). Course of chitin production and subdivision of stage 16: As detected by the chitin-binding dye Fluostain, compared to early stage 16 (16a, A), in addition to the tracheal plexus that contains chitin (tra), the head skeleton (hs) at late stage 16 (16b, B) also produces chitin. Subsequently, at early stage 17 (17a, C), the epidermis starts to produce chitin (arrow in C). Interestingly, at early stage 16, chitin can be detected in the hindgut (*). Embryos of stage 16a and 16b are both hardly visible in cuticle preparations (D,E). Subdivision of stage 17: At early stage 17 (17a, F), the pharynx emerges (arrows) and the surface of the embryo smoothens. Stage 17b is defined by the emergence of the entire head skeleton, and the epidermal cuticle clearly lines the body of the embryo (G). In the living embryo, the dorsal trunk (tra) and the Malpighian tubules (mal) become visible (J). The labrum (arrow) and the ventral denticles (triangle) start to melanise at stage 17c (H). As seen in the living animal, the dorsal trunk is now air-filled (K). Finally, at stage 17d the head skeleton is entirely melanised (I,L). (AeC,L) dorsal view and (DeK) lateral view of representative embryos. Anterior is to the left.
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Fig. 3. Ultrastructure of epidermal cells of embryos at stages 11 to 14. At stages 11 to 14 (AeD) the epidermal cells are columnar, and contain a well-developed ER network and many lipid drops. Mitochondria are often found at the apical portion of the cell. At stage 14 (D), the epidermal cells accumulate glycogen. In stages 11 (E) to 14 (F) microtubules run parallel to the apical plasma membrane, and a membranous structure called the first embryonic cuticle covers the embryos. aj, adherens junction; EC1, embryonic cuticle 1; er, endoplasmatic reticulum; gl, glycogen; ld, lipid drops; mt, microtubules; n, nucleus; vm, vitelline membrane. Scale bars 500 nm, scale bar in A applies for AeD.
(ENZO Life Sciences, Farmingdale, USA) and protein A10 nm gold conjugates (gift from Dr. York Stierhof, ZMBP Tu¨bingen). Labelled sections were then stained with 1% aqueous uranyl acetate for 3 min and lead citrate. For scanning electron microscopy embryos were fixed for five hours in 4% formaldehyde and 0.5% glutaraldehyde at 4 C. They were manually devitellinized in PBS, osmium treated (1% osmium tetroxide in 100 mM phosphate buffer, pH 7.2), dehydrated through an ethanol series, subjected to critical point drying from CO2 and sputter coated with 10 nm Au-Pd. Samples were examined at 20 kV accelerating
voltage in a Hitachi S800 field emission scanning electron microscope. 3. Results 3.1. Subdivision of stages 16 and 17 Cuticle differentiation as described by Hillman and Lesnik (1970) occurs during the commonly employed embryonic stages 15, 16 and 17 of Campos-Ortega and Hartenstein (1985). According to the latter publication, the two terminal
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Fig. 4. Ultrastructure of the differentiating cuticle at stage 15. Epidermal cells are more compact during stage 15 than in the previous stages (A). At the tips of irregular membrane protrusions fragments of the envelope are formed (B). Larger fragments of the envelope are formed at denticles (B 0 ). EC1, embryonic cuticle 1; ed, ecdysal droplets; env, envelope; gl, glycogen; mt, microtubules; n, nucleus; rif, plasma membrane riffles; vm, vitelline membrane. Scale bars in A and B 500 nm, in B 0 250 nm.
stages 16 and 17 last for about 11 h (13 to 24 h after egg laying, AEL). To have a finer staged series to standardise embryo collection for electron microscopy, we subdivided stages 16 and 17 monitoring the course of cuticle differentiation, chitin production and cuticle melanisation (tanning) on fixed and live animals by light and fluorescence microscopy. Differentiation of other morphological landmarks such as the tracheal system, the midgut and the Malpighian tubules is included to facilitate rapid staging. The data are summarised in Fig. 2 and Table 1. At stage 16 (13 to 16 h AEL, Figs. 2A,B,D,E), the embryo is completely removed by Hoyer’s medium and no cuticle is recognisable in cuticle preparations; however, chitin as a marker for the procuticle, is detectable in the head skeleton at the last third of this stage (15 h AEL). We use this criterion to subdivide stage 16 in stages 16a (13 to 15 h AEL) and 16b (15 to 16 h AEL). At the beginning of stage 17 (16 to 17 h AEL, Figs. 2C,F) the epidermal cuticle, albeit thin, and the pharynx emerge in cuticle preparations. As previously shown (Moussian et al., 2005a), chitin is detected in the epidermis and strongly in the ventral
denticles at this stage (not shown). Other striking differences between stage 16 and stage 17 embryos are the constriction of the midgut lobes and the appearance of the Malpighian tubules, both observed on living animals (not shown). Based on these differences, we call this period stage 17a. Subsequently, the dorsal trunk, albeit not yet air-filled, becomes easily discernable from the surrounding tissue (stage 17b, 17 to 18.25 h AEL, Figs. 2G,J). The head skeleton and ventral denticles remain un-melanised, but are now clearly visible. At the beginning of stage 17c, the dorsal trunk starts to fill with air, and at the end of this stage the whole tracheal plexus is air-filled (18.25 to 19 h AEL, Figs. 2H,K). Concomitantly, the ventral denticles and the labrum begin to melanise, and the Malpighian tubules darken. Finally, at stage 17d, the entire head skeleton melanises, and the animal starts to move within the egg case (19 to 22.5 h AEL, Figs. 2I,L). Embryos start to hatch 20.5 h AEL, and over three quarters of the embryos have hatched after 22.5 h AEL. To study cuticle differentiation, thin sections of cryo-immobilized embryos from stage 11 to the end of embryogenesis (including the defined sub-stages 16a and b and 17aed),
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Fig. 5. Ultrastructure of the differentiating cuticle at stage 16a. Fragments of the envelope separated by small gaps (open triangles) cover almost the entire surface of the epidermal cells that are cuboidal now (A). By contrast, the envelope is continuous at the denticles that start to form a premature electron-dense epicuticle (A 0 ), which is absent at the naked cell surfaces (B,C). As shown on cross (B) and longitudinal sections (C), the apical plasma membrane forms extended protrusions that we call apical undulae (see also Fig. 15). aj, adherens junction; bl, basal lamina; d, denticle; env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; mt, microtubules; n, nucleus; pro, procuticle; und, apical undulae; vm, vitelline membrane. Scale bars in A, B and C 500 nm, in A 0 250 nm.
were examined using a transmission electron microscope (TEM). In addition, we detected chitin by gold-labelled WGA on thin sections to follow procuticle assembly. 3.2. Cuticle differentiation from stage 11 to 14 The epidermal cells are columnar at stages 11 to 14 (Figs. 3AeD), and the nucleus lies in the middle of the cell. They are connected via adherens junctions; typical ladder-like
septate junctions, by contrast, are missing. The cytoplasm contains many ER cisternae and mitochondria that preferentially localise to the apical portion of the cell, underneath a zone of clear cytoplasm, which harbours only microtubules that are oriented in parallel to the apical plasma membrane (Figs. 3E,F). Microtubules are also abundant at the lateral plasma membrane in an apical-basal orientation. Occasionally glycogen accumulates basally; a basal lamina is not present.
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Fig. 6. Ultrastructure of the differentiating cuticle at stage 16b. The epidermal cells are cuboidal, and the fragments of the envelope have fused to a continuous layer (A). The epicuticle is now being formed also underneath the envelope of the naked cell surfaces (B). d, denticle; ed, ecdysal droplets; env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; mt, microtubules; n, nucleus; pro, procuticle; vm, vitelline membrane. Scale bars 500 nm.
Neither chitin is produced nor cuticle is formed at these stages, however instead, an irregular thin sheath covers the embryo. The space between the sheath and the epidermal surface contains only a small amount of disordered material (Figs. 3E,F). This structure was not described by Hillman and Lesnik (1970), but had been found in other dipteran embryos and may be homologous to the so-called first embryonic cuticle (EC1) observed in many insects (Truman and Riddiford, 1999).
of the riffles fragments of the outermost envelope are eventually produced. Ventrally, primordia of denticles containing cables of microtubules and probably actin filaments are emerging. Their apical plasma membrane remains smooth, however, the envelope is almost continuous at these positions (Fig. 4B 0 ). The appearance of the denticles is earlier than determined by cuticle preparations and light microscopy.
3.3. Cuticle differentiation at stage 15
Stage
Envelope
Epicuticle
Procuticlea
ECSb
Cell shape
15 16a 16b 17a 17b 17c 17d
<10 nm <10 nm <10 nm 10 nm 15 nm 20 nm 20 nm
e e 10 nm 40 nm 70 nm 120 nm 120 nm
e 25 nm 30 nm 80 nm 160 nm 300 nm 300 nm
85 nm 110 nm 130 nm 135 nm 240 nm 460 nm 460 nm
Columnar-cuboidal Cuboidal Cuboidal Cuboidal Cuboidal-flat Flat-cuboidal Flat
At stage 15, cells at the dorsal side of the embryo start to flatten (Fig. 4A), but are still columnar, whereas ventral cells remain as they were at previous stages (not shown). The nucleus moves to the basal side of the cell, where glycogen further accumulates. The cytoplasm is organised as in previous stages. The apical plasma membrane roughens irregularly (Figs. 4A,B). The apical microtubules run at the basis of these structures parallel to the cell surface. This is the stage at which cuticle differentiation starts. At the tips
Table 2 Growth of the cuticle during stages 15 to 17d
Until stage 17c, growth is continuous, therefore average thicknesses are given. a The procuticle is defined as the chitin-harbouring layer. b The extracellular space (ECS) is the widest distance between the apical plasma membrane and the surface of the envelope.
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Fig. 7. Ultrastructure of the differentiating cuticle at stage 17a. The epidermal cells are cuboidal (A). The epicuticle is now partitioned into a lower electron-dense and an upper electron-lucid sub-layer (B,C). Apical undulae are still present as observed in cross (B) and longitudinal sections (C). aj, adherens junction; env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; mt, microtubules; n, nucleus; pro, procuticle; und, undulae. Scale bars 500 nm.
No chitin is detected at this stage (Fig. 8A), and the EC1 is intact. 3.4. Cuticle differentiation at stage 16a All epidermal cells are now cuboidal (Fig. 5A). The organisation of the cytoplasm is unchanged. The nucleus is located to the basal half of the cell. The basal lamina starts to be produced. The apical plasma membrane now forms regular microtubule-containing corrugations that can be seen in longitudinal but not in cross
sections (Figs. 5B,C). Hence, both these corrugations and their microtubules run perpendicular to the anteroposterior axis of the animal. We name these structures apical undulae. They are not formed at the plasma membrane of denticle buds. For the first time, chitin is detected by gold-labelled WGA underneath the almost continuous envelope (Fig. 8B). It is, however, not possible to discern any chitin laminae (Figs. 5B,C). Obviously, detection of chitin on thin sections is more sensitive than on fixed embryos (stage 17a, Fig. 2C). There is no sign of epicuticle formation except beneath the
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envelope of denticles (Fig. 5A 0 ). The EC1 dissociates, and electron-dense droplets reminiscent of ecdysal droplets that are formed during moulting, are present apically outside the cell. In total, the EC1 covers the embryo for about five to seven hours. Interestingly, it persists during epidermal morphogenesis (dorsal closure and head involution) from stage 14 to 15 indicating its flexibility. 3.5. Cuticle differentiation at stage 16b Epidermal cells are cuboidal, and their cytoplasm remains organised as at the previous stage (Fig. 6A). The apical undulae persist (not shown), the amount of glycogen slightly diminishes, whilst procuticle formation continues (Figs. 6B and 8C, Table 2). Chitin laminae are not detectable morphologically. The electron-dense epicuticle is now seen beneath the envelope of all epidermal cells, while the electron-lucid epicuticle is not formed yet (Fig. 6B). 3.6. Cuticle differentiation at stage 17a Epidermal cells and their contents are indistinguishable from cells at stage 16b (Fig. 7A), the apical undulae are present (Figs. 7B,C). The electron-lucid epicuticle is formed between the thin electron-dense epicuticle and the envelope. Hence, at this stage, the cuticle has roughly the organisation of the mature cuticle. The procuticle thickens due to a continuous secretion of chitin (Figs. 7B,C and 8D, Table 2); chitin laminae, however, remain indiscernible. 3.7. Cuticle differentiation at stage 17b At stage 17b, epidermal cells flatten (Fig. 9A), the lateral membrane undulates, and septate junctions form ladder-like arrays. The cytoplasm is predominantly occupied by glycogen and ER cisternae. Lateral microtubules persist, but less microtubules run parallel to the surface membrane. Apical undulae are now flatter and irregularly spaced (not shown). Due to a broader epicuticle that is now clearly distinguishable from the other two layers, in addition to chitin synthesis, the cuticle thickens substantially (Figs. 9B,C and 8E, Table 2). However, chitin laminae are not visible. As observed in many other insects, chitin seems to be absent from the epicuticle. Electron-dense tube-like structures, probably precursors of the pore canals, are formed in the epi- and the procuticle (Figs. 9B,C). At late stage 17b a third electron-dense sheet is added to the envelope. 3.8. Cuticle differentiation at stage 17c Fig. 8. Detection of chitin by gold-labelled WGA (black dots in the procuticle, pro, brackets) on thin sections for TEM reveals that the formation of the procuticle starts after the onset of envelope formation at stage 15 (A) during stage 16a (B), and before the epicuticle emerges at stage 16b (C). During the stages 17a (D), 17b (E) and 17c (F) chitin amount in the procuticle increases. At stage 17d (G), chitin production seems to have ceased, as the procuticle does not grow further. Alternatively, a tighter packaging of newly produced chitin into laminae without changing procuticle thickness may mask chitin production. Scale bar in A is 500 nm and applies for AeG.
No major changes are observed in the organisation of epidermal cells at this stage compared to cells at stage 17b (Figs. 9A and 10A). The amount of glycogen decreases, and apical undulae have almost vanished (Fig. 10D). The layers grow (Table 2), chitin amount increases but laminae are still not visible (Figs. 10BeD and 8F). The lumen of the pore canals broadens (inset in 10C), and the mature envelope is
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Fig. 9. Ultrastructure of the differentiating cuticle at stage 17b. The epidermal cells are flatter than at the previous stage (A), and the lateral membranes lengthen and become wavy (arrows). Since stage 17a (Figs. 7B,C), the procuticle has thickened remarkably (B,C). The envelope maturates to a five-lamellar structure, and pore canals start to form (B,C). To visualise chitin laminae that have a helicoidal arrangement giving a parabolic texture to the procuticle, cross (B) and oblique sections (C) are compared. No chitin laminae can be seen at this stage. env, envelope; epi, epicuticle; gl, glycogen; hl, haemolymph; n, nucleus; pc, pore canal; pro, procuticle. Scale bars 500 nm.
now clearly composed of five alternating electron-dense and electron-lucid sheets. 3.9. Cuticle differentiation at stage 17d At the end of stage 17d the apical plasma membrane of the flat cell (Fig. 11A) is devoid of apical undulae (not shown) and epidermal cells cease to secrete cuticle material, as ER cisternae are almost absent, suggesting that the larva is ready to hatch. The basal lamina maturates (compare to Fig. 5A). Glycogen has been entirely consumed, and the amount of chitin does not seem to change compared to the previous stage (Fig. 8G, Table 2). Chitin laminae are now fully differentiated and condensed (Figs. 11B,C). They gain their typical arrangement that gives the procuticle a parabolic texture (Bouligand, 1965) (see also Fig. 1). The arrangement of the upper half of the procuticle changes in mature ventral denticles and is rather spongy (Fig. 12A). In addition, the electron-dense epicuticle broadens
and further darkens in denticles; this probably reflects sclerotisation and pigmentation observed in cuticle preparations (Fig. 2I). However, stage 17d embryos mutant for Ddc with defects in sclerotisation and pigmentation (Wright, 1987) exhibit nevertheless darkened denticular epicuticle (Fig. 12B). Hence, sclerotisation and pigmentation are not unambiguously observable with an electron microscope. The gross morphology of denticles in chitin deficient cuticles from krotzkopf verkehrt (kkv) mutant embryos is normal (Figs. 12D,E) suggesting that chitin and the procuticle, which is almost missing in kkv mutant embryos (Moussian et al., 2005a) (Fig. 12C) are not required for shaping a denticle. 3.10. Formation and degradation of tracheal luminal chitin The tracheal luminal chitin filament has been shown to assist tracheal tube dilation during embryogenesis (Tonning et al., 2005). To verify staging of the embryos sectioned for
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Fig. 10. Ultrastructure of the differentiating cuticle at stage 17c. The epidermal cells are flat (A). The cuticle continues thickening during this stage, but no chitin laminae can be discerned in oblique (C) compared to cross sections (B). Pore canals (*) are clearly distinguishable form the surrounding cuticle (inset in C). Only shallow traces of the apical undulae are seen in longitudinal sections (D). env, envelope; epi, epicuticle; gl, glycogen; ld, lipid drops; n, nucleus; pc, pore canal; pro, procuticle; und, undulae; vm, vitelline membrane. Scale bars in AeD 500 nm, in the inset of C 250 nm.
TEM analysis we monitored the course of tracheal luminal chitin formation and degradation (Fig. 13). From stages 15 to 16b (Figs. 13AeD), the lumen of the developing trachea contains chitin fibres running parallel to the length of the tracheae. At stage 17a, electron-lucid areas emerge in-between the fibres that seem to lose their continuity (Fig. 13E). At stage 17b, only little material is left in the lumen of the tracheae (Fig. 13F). Finally, at stage 17c, the lumen of the tracheae is cleared; this is the stage when tracheae fill with air (Fig. 2K
and not shown). These observations differ slightly from data published recently by Anna Tonning and colleagues in Developmental Cell in 2005, insofar as degradation of chitin occurs later in our TEM study compared to their analysis on whole mount embryos. Interestingly, luminal chitin is removed or absorbed when the envelope entirely covers the apical surface of the cells indicating that a mechanism must exist, which allows transport of conceivably degraded chitin molecules through the differentiating tracheal cuticle.
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Fig. 11. Ultrastructure of the differentiating cuticle at stage 17d. The epidermal cell has further flattened, and does not contain any glycogen (A). The cuticle ceases thickening at this stage, when orientation of chitin laminae takes place as observed in oblique (C) sections compared to cross sections (B). Pore canals persist (inset in B). The apical plasma membrane of the epidermal cell is flat, and apical undulae are absent. bl, basal lamina; env, envelope; epi, epicuticle; hl, haemolymph; n, nucleus; pc, pore canal; pro, procuticle. Scale bars 500 nm, in the inset of B 250 nm.
4. Discussion The Drosophila larval cuticle, a three-layered aECM, is produced in the second half of embryogenesis. The events taking place during cuticle differentiation are summarised in Fig. 14 and Table 2. 4.1. Summary of cuticle differentiation 4.1.1. Embryonic cuticle 1 Prior to cuticle formation in the truest sense of the word there is an aECM that is most probably homologous to the so-called first embryonic cuticle (EC1) found in many insects (Truman and Riddiford, 1999). The function of the EC1 is still elusive. In Drosophila, it may be an evolutionary relict as in more basal insects such as Therombia domestica (silverfish) the EC1 still contains fibrous material (chitin?). In the Drosophila embryo, this structure is free from chitin.
Alternatively, as proposed by Tepass and Hartenstein (1994), it may be just the extracellular matrix that exists before cuticle differentiation. In this sense, the term ‘‘cuticle’’ may be misleading. 4.1.2. The envelope The envelope is the outermost layer of the cuticle and protects the animal from dehydration. Its precursor, consisting of two electron-dense sheets enclosing an electron-lucid one, is produced at the tips of plasma membrane riffles at stage 15, and the gaps between the fragments are closed by a yet unknown mechanism at stages 16a and 16b. No changes are observed during stages 17a and 17b, but at the transition of stage 17b to 17c, a third electron-dense sheet is formed within the electron-lucid one. The mature envelope has five alternating electron-dense and electron-lucid sheets. This is the same arrangement as observed in the envelope (‘‘cuticulin layer’’) of the Calpodes ethlius larva by Locke (1966).
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observed at stage 16b. The bulk of chitin, however, is secreted in a short time period at the stages 17b and 17c. Hence, chitin accumulation within the extracellular space seems not to be continuous, indicating a peak of chitin synthase activity at these stages. The typical parabolic texture of chitin laminae becomes visible only at stage 17d, prior to hatching. 4.2. Phases of cuticle differentiation
Fig. 12. The procuticle is not needed for denticle morphology. As seen in cross sections of the wild-type denticle, the centre of the denticle harbours the brighter extension of the procuticle that seems to be continuous with the electron-dense epicuticle, which directly contacts the envelope (A). In the Ddcn7 mutant denticle the epicuticle appears to be normal (B). The denticle of kkvIB22 mutant embryos the epicuticle is clearly separated from the remnants of the procuticle (C). The wild-type ventral denticle has a characteristic shape with the bent tip pointing to the posterior (D). The denticle of the chitin-deficient kkvIB22 mutant embryo has an identical shape (E). Note that the surface of the kkvIB22 mutant embryo is wrinkled due to the defective procuticle. Scale bars in AeC 500 nm, in D and E 1 mm.
4.1.3. The epicuticle The epicuticle emerges first at stage 16b underneath the closed precursor of the envelope. At stage 17a, it is partitioned into an upper electron-lucid and a lower electron-dense sublayer. Thereafter, it thickens continuously. At stage 17b, pore canals that yet lack a lumen are established within the epicuticle. At stage, 17c, the pore canals expand to form a lumen. 4.1.4. The procuticle Secretion of chitin to form the procuticle starts during envelope assembly at stage 16a and before epicuticle secretion can be
In summary, we propose that cuticle differentiation in the Drosophila embryo may be divided into three phases (Fig. 14). First, during the phase of establishment (stage 15 to 17a), the envelope, the procuticle (chitin) and the epicuticle are established and the layers adopt their final shape. Interestingly, the lower procuticle starts to be secreted at stage 16a before the middle epicuticle emerges at stage 16b. Next, at stages 17b and 17c, during the phase of growth the epicuticle and the procuticle substantially thicken. Finally, at stage 17d, the cuticle acquires its physical qualities as chitin laminae of the procuticle gain their typical orientation and the head skeleton and the ventral denticles melanise. The course of cuticle differentiation in the Drosophila embryo described here differs in some substantial points from that published by Hillman and Lesnik (1970). In their work, they show that cuticle differentiation is sequential, the outermost envelope being produced as the first, and the innermost procuticle as the last layer. Moreover, we show that the envelope prior to hatching is five-lamellar rather than tri-lamellar as it is in the larva of Calpodes ethlius (Locke, 1966). Our description of cuticle establishment also differs from that described for Manduca sexta embryos (Ziese and Dorn, 2003), which is very similar to that of Drosophila presented by Hillman and Lesnik (1970). We think that the differences mainly derive from the different fixation techniques affecting specimen preservation. Cryo-immobilisation followed by freezesubstitution reflects the natural state of the embryo better than direct chemical fixation followed by dehydration at
Fig. 13. Tracheal cells, as shown here for the posterior cells of the dorsal trunk, produce a luminal chitinous filament that is required for tube diameter regulation. At early stage 15 (A), luminal chitin fibres have a wavy appearance and are in close contact to the surface of the tracheal cells that form fragments of cuticular envelope (env) at late stage 15 (B). At stages 16a and b, the envelope is continuous, the chitin filament is unchanged (C,D). At stage 17a, electron-lucid patches separate the chitin fibres (E). At stage 17b, almost no chitin fibres are found in the tracheal lumen that is now almost devoid of any electron-dense material (F). env, envelope; lum, tracheal lumen; tae, taenidial fold. Scale bars 500 nm.
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room temperature. Moreover, we traced back procuticle establishment with gold-labelled WGA detecting chitin, which is more sensitive than judging upon morphological features only. Alternatively, differences in ecological strategies and life cycles of Manduca sexta (120 h of embryogenesis) and Drosophila melanogaster (22.5 h of embryogenesis) may impose different mechanisms of cuticle production.
env
rif
stage 15 env
pro (chitin)
und
Phase 1
stage 16a pro (chitin)
4.3. The undulae
epi
stage 16b
The apical plasma membrane protrudes riffles at stage 15 at the tip of which the envelope is formed. Thereafter, between stage 16a and 17b the epidermal cells form apical corrugations, the apical undulae, that are stabilised by microtubules (Fig. 15). They are arranged in an angle of 90 degree to the length of the embryo, and chitin microfibrils adjacent to the cell surface are formed perpendicular to them. As they contain microtubules they are different from the taenidial folds of the trachea (Figs. 13CeF) that lack microtubules but are fortified by actin filaments (Matusek et al., 2006). We do not find any similar cellular structure in the literature, and we suspect that they play a stabilising role before the cuticle assumes this function. In addition, they may be required for correct orientation of microfibrils that are placed in a right angle to the undulae. 4.4. Cuticle differentiation in denticles
stage 17a
Phase 2
stage 17b/c
cross
long
stage 17d
cross
Phase 3
long
envelope epicuticle procuticle Fig. 14. Model of the three phases of cuticle differentiation in the Drosophila embryo. First, the three layers are established from stage 15 to stage 17a (phase 1 or phase of establishment). Microfibrils (red lines) that are produced perpendicular to the apical undulae are assembled into laminae. Second, the cuticle thickens (phase 2 or growth phase). The uniform orientation of the microfibrils within the laminae is shown in a cross section (cross) of the longitudinal view (long). Third, the chitin laminae rotate (phase 3 or phase of maturation). At the same time the ventral denticles and the head skeleton darken. In each lamina the portion of a single microfibril that lies in the plane of the drawing is highlighted in dark red. The rotation of the microfibrils is demonstrated in the cross section of the longitudinal view: in the extreme cases, a cross-sectioned microfibril in the longitudinal view appears longitudinal in the cross section and vice-versa. env, envelope; epi, epicuticle; pro, procuticle; rif, riffles; und, undulae.
The cuticle of denticles is thicker than the cuticle of naked cell surfaces, suggesting that different cellular mechanisms of cuticle differentiation may occur at these positions. Indeed, riffle and undulae formation is suppressed at those sites of the apical plasma membrane where denticles emerge, and the electrondense plaques underline the entire denticle producing plasma membrane. This suppression may enlarge the area of secretion of proteins as well as increase the density of chitin synthase machinery. Hence, riffles and undulae in epidermal cells, in contrast to the situation in gut epithelial cells, may probably not form to enlarge the secretory surface. Another speciality of the denticles is that their melanisation requires the localisation of melanisation enzymes to the denticles, prohibiting their spreading. There is no evidence how this is achieved. 4.5. Some molecular aspects of cuticle differentiation The non-sequential manner of secretion and layer-organisation implies that extracellular cuticle components to some extent possess a self-organising capacity. Namely orientation of chitin laminae seems not to occur during chitin synthesis and microfibril formation at the apical plasma membrane, but later at stage 17d when the bulk of chitin and cuticle proteins have already been deposited in the extracellular space. Similarly, self-assembling processes have been suggested to govern the formation of alternating dense and watery layers within the procuticule of butterfly pupae (Steinbrecht, 1985). Hence, in the Drosophila embryo, procuticle maturation proceeds in two steps. First, at stages 15 to 17c, chitin microfibrils are formed and arranged
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undulae
A
151
B aj
nucleus longitudinal section
cro ss se cti on
mt
aj
B’
chitin
mt
und
D
C chitin
und mt
mt
und
chitin
Fig. 15. The apical undulae. Model of the apical undulae that are formed during stages 16a to 17b at the surface of epidermal cells (A). Longitudinal section of an embryo at stage 17a shows the apical undulae (und) as regular protrusions of the apical plasma membrane (B and B 0 ). Tangential section of the surface of an embryo at stage 17a reveals that chitin microfibrils run perpendicular to the apical undulae (C). Oblique-longitudinal section of an embryo at stage 17a demonstrates that long strands of microtubules (mt) underlay the apical undulae (D). aj, adherens junction; mt, microtubule; und, undulae. Scale bars in B, C and D 1 mm, in B 0 500 nm.
in parallel at the apical plasma membrane, processes that, as suggested by the mutant phenotypes (Moussian et al., 2005b, 2006), require the function of the membrane-bound proteins Knickkopf (Knk) and Retroactive (Rtv). Second, chitin laminae rotate with respect to each other at stage 17d, probably through an interaction with extracellular chitin-binding proteins. Assembly of chitin microfibrils and laminae is also influenced by the secreted chitin deacetylases Serpentine (Serp) and Vermiform (Verm) that presumably remove the acetyl-group from the chitin monomer N-acetyl-glucosamine thereby changing the physical properties of chitin (Luschnig et al., 2006). Likewise, in plants, physical as well as enzymatic reactions in the extracellular space have been proposed to be responsible for organising the network of cellulose and other polysaccharides during cell wall differentiation (Cosgrove, 2005). We are still a long way towards an understanding of the molecular mechanisms of cuticle assembly. Notably the
mechanisms of envelope and epicuticle differentiation are enigmatic. Based on the present study, the characterisation of phenotypes caused by mutations in genes coding for cuticle proteins contributes to our learning of how the insect cuticle as an example of an aECM is assembled. Acknowledgements We would like to thank Christiane Nu¨sslein-Volhard for support, and Matthew Harris for critical reading of the manuscript. References Bouligand, Y., 1965. On a twisted fibrillar arrangement common to several biologic structures. Comptes rendus he´bdomadaires des se´ances de l’Acade´mie des sciences. Se´rie D: Sciences Naturelles 261, 4864e4867.
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Campos-Ortega, J.A., Hartenstein, V., 1985. The Embryonic Development of Drosophila melanogaster. Springer Verlag, New York. Cosgrove, D.J., 2005. Growth of the plant cell wall. Nature reviews. Molecular Cell Biology 6, 850e861. Guthrie, D.M., Tindall, A.R., 1968. The Biology of the Cockroach. Edward Arnold Ltd., London. Hillman, R., Lesnik, L.H., 1970. Cuticle formation in the embyro of Drosophila melanogaster. Journal of Morphology 131, 383e396. Locke, M., 1966. The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae). Journal of Morphology 118, 461e494. Locke, M., 1969. The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid in an insect. Journal of Morphology 127, 7e39. Locke, M., 2001. The Wigglesworth Lecture: insects for studying fundamental problems in biology. Journal of Insect Physiology 47, 495e507. Luschnig, S., Batz, T., Armbruster, K., Krasnow, M.A., 2006. serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila. Current Biology 16, 186e194. Matusek, T., Djiane, A., Jankovics, F., Brunner, D., Mlodzik, M., Mihaly, J., 2006. The Drosophila formin DAAM regulates the tracheal cuticle pattern through organizing the actin cytoskeleton. Development 133, 957e966. McDonald, K., Morphew, M.K., 1993. Improved preservation of ultrastructure in difficult-to-fix organisms by high pressure freezing and freeze substitution: I. Drosophila melanogaster and Strongylocentrotus purpuratus embryos. Microscopy Research and Technique 24, 465e473. Moussian, B., Schwarz, H., Bartoszewski, S., Nu¨sslein-Volhard, C., 2005a. Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster. Journal of Morphology 264, 117e130. Moussian, B., So¨ding, J., Schwarz, H., Nu¨sslein-Volhard, C., 2005b. Retroactive, a membrane-anchored extracellular protein related to vertebrate snake neurotoxin-like proteins, is required for cuticle organization in the larva of Drosophila melanogaster. Developmental Dynamics 233, 1056e1063.
Moussian, B., Tang, E., Tonning, A., Helms, S., Schwarz, H., Nu¨ssleinVolhard, C., Uv, A.E., 2006. Drosophila Knickkopf and Retroactive are needed for epithelial tube growth and cuticle differentiation through their specific requirement for chitin filament organization. Development 133, 163e171. Mu¨ller, M., Moor, H., 1984. Cryofixation of thick specimens by high pressure freezing. In: Revel, J.-P., Barnard, T., Haggis, G.H. (Eds.), The Science of Biological Specimen Preparation for Microscopy and Microanalysis, vol. Proeceedings of the second Pfefferkorn Conference at the Sugar Loaf Mountain Resort. Scanning Electron Microscopy, Inc., Traverse City, MI. AMF O’Hare, IL 60666, pp. 131e138. Neville, A.C., 1975. Biology of the Arthropod Cuticle. Springer Verlag, Berlin Heidelberg New York. Steinbrecht, R.A., 1985. Fine structure and development of the silver and golden cuticle in butterfly pupae. Tissue and Cell 17, 745e762. Studer, D., Michel, M., Mu¨ller, M., 1989. High pressure freezing comes of age. Scanning Microscopy, Supplement 3, 253e268; discussion 268e269. Tepass, U., Hartenstein, V., 1994. The development of cellular junctions in the Drosophila embryo. Developmental Biology 161, 563e596. Tonning, A., Hemphala, J., Tang, E., Nannmark, U., Samakovlis, C., Uv, A., 2005. A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea. Developmental Cell 9, 423e 430. Truman, J.W., Riddiford, L.M., 1999. The origins of insect metamorphosis. Nature 401, 447e452. Wieschaus, E.F., Nu¨sslein-Volhard, C., 1986. Looking at embryos. In: Roberts, D.B. (Ed.), Drosophila: A Practical Approach. IRL Press, Washington, D.C., pp. 199e227. Wright, T.R., 1987. The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. Advances in Genetics 24, 127e222. Ziese, S., Dorn, A., 2003. Embryonic integument and ‘‘molts’’ in Manduca sexta (Insecta, Lepidoptera). Journal of Morphology 255, 146e161.