Stage and tissue-specific expression of a collagen gene during Drosophila melanogaster development

Experimental

Cell Research 163 (1986) 405-412

Stage and Tissue-specific Expression of a Collagen Gene during Drosophila melanogaster Development Y. LE PARCO, B. KNIBIEHLER, Laboratoire

J. P. CECCHINI

and C. MIRRE

de Biologie de la Diffhenciation Cellulaire, FacultC des Sciences de Marseille-Luminy, 13288 Marseille Cedex 9, France

Based on data from developmental RNA profiles and in situ hybridization, we report a direct examination of the expression of one collagen gene (Dcgl) during Drosophila melanogaster life cycle. These studies show, for the first time, that the expression of a collagen gene is both differential and tissue-specific during the course of development. Moreover, they demonstrate that the connective tissues in Drosophila do contain a collagen type synthesized by mesodermal tissues. Indeed the accumulation of Dcgl transcripts was located mainly within the second instar fat bodies, the third instar lymph glands, and over adepithelial cells associated with third instar imaginal discs. In addition, these results seem to confirm the interpretation that wandering hemocytes released by the lymph glands could contribute in extracellular matrix composition in some tissues in the kilTa.

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1986 Academic

Ress,

Inc.

The collagenous proteins occur as a family of molecules that has been detected in every metazoa phyla. These proteins have an important structural function in the extracellular matrix which surrounds the cells, but they also perform a key role in the control of tissue morphogenesis during embryonic development. The number of known collagenous proteins is continuously expanding, but it is now clear that they belong to several genetically distinct types that have evolved for a specific function outside the cells [l]. In this respect, the study of collagenous proteins in insects in general and in Drosophila has a phylogenetic significance. Indeed the collagenous connective tissues are widespread in insects, but, due to the presence of an exoskeleton and of large amounts of chitin, their scarcity was a barrier in their study. In consequence, the presence of collagens in insects has been evidenced mainly by ultrastructural studies [2]. Their extent, exact nature, processes of formation and functions are still far from certain. In order to elucidate these features, a good way is to isolate and characterize, within a given species, most of collagen genes and to assess the time of appearance and the tissue distribution of their products during the course of development. We thus have undertaken an exhaustive molecular study of the collagen gene family in Drosophila melanogaster [33]. Today, we have cloned ten ‘collagenlike’ genes, one of them being the Dcgl collagen gene previously described by Monson et al. [3, 43. Copyright @ 1986 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/86 $33.00

406 Le Parco et al.

Fig. 1. (A) The-r$dioactive Pcg45 probe was hybridized to restriction digests of Drosophila genomic clone Dcgl to determine the regions of homology between chicken and Drosophila sequences. The Pcg45 inset was hybridized to filters in low stringency conditions (50% formamide: SxSSC, 37”C, 24 h) and the filters were washed in 0.5xSSC at 42°C. B, BamH,; E, EcoRi; H, HindHI. (B) Restriction maps of the three overlapping Drosophila collagen clones. The three clones Dcgl, Dcgl4 and Dcg27 are localized on polytene chromosomes to the 2L-25C locus [33]. Boxes represent regions of Drosophila DNA that cross-hybridize, highly (U) or slightly (Cl) to the “P-nick-translated Pcg45 probe. Abbreviations as in (A). 0, Dcg [3]. (C) Genomic hybridization patterns generated by the BamHr-EcoR, (BE) fragment (B, n ). The genomic DNA from Drosophila OreR strain was digested to completion either with Hind111 (H), or, EcoRr-Hind111 (EH), or, BamHr-EcoRr (BE’). The fragments were separated on 1% agarose gel and transferred to nitrocellulose filters. Each lane contained 10 pg DNA. The BE fragment was labelled by nick translation and hybridized to the tllter at 42°C in 50% formamide: IxSSC, 0.1% SDS, 20 mM mercaptoethanol, 24 h. The filter was washed at 42°C in O.SxSSC. The mobilities of HindIII-restricted phage DNA were used as size markers.

The results presented here are based on developmentalRNA profiles and on in situ hybridization of Dcgl collagen DNA probe over frozen sections of all Drosophila developmental stages. They allowed us to define the exact timing of expression of this gene and threw a new light on the contention as to the cellular origin of connective tissues in Drosophila. DNA Procedures

MATERIAL AND METHODS

The methods used for phage DNA preparation, agarose gel electrophoresis and Southern blot hybridizations, were essentially performed as described in Maniatis et al. [5]. The specific conditions for hybridization were as indicated in the caption of fig. 1. The DNA transfer procedures on Exp Cell Res 163 (1986)

Drosophila collagen gene expression 407

Fig. 2. (A) Developmental poly(A+) mRNA profiles of Dcgl expression. 5 pg was deposited on each

lane. The conditions of hybridization were the same as in fig. 1c. The developmental stages represented are: I, t&20 h embryos; 2-5, fust, second, early and late third-instar larvae; 6, white pupa; 7-9, early to late pupa; 10, adult insect; 11, KC0 Drosophila cells. (B) Northern blot analysis of some particular stages was performed using a BE fragment labelled with the four 32Pdeoxynucleotides. Each lane contained 10 pg poly(A+) mRNA. Conditions were the same as in fig. 1c. Lanes I’, 4’, 7’ and 10’ correspond to lanes I, 4, 7 and 10 in (A).

nitrocellulose filters were according to Kafatos et al. [6]. For restriction enzyme analysis, the samples of DNA were dissolved in 10 mM ‘Iris-HCI (pH 8), 1 mM EDTA, and digested with enzymes (3-4 units/ug DNA) at 37°C for 1 h in appropriate buffers [5]. The isolation and purification of a given restriction fragment were performed as follows: the restriction digest of recombinant phage DNA was size-fractionated on a 1% low melting point agarose gel (BRL) and run with ethidium bromide (EB). The agarose slice containing the fragment was excised under long-wave UV light and melted at 65°C for 10 min in an equal volume of 10 mM ‘Bis-HCl (PH 8), in 1 mM EDTA, then it was extracted three times at 37°C with phenol, washed three times with ether and ethanol-precipitated (30 min at -8O’C).

Isolation of RNA and Northern Blot Analysis The development stages of Drosophila were stockpiled in liquid nitrogen. The total RNAs were isolated by the phenol-chloroform procedure as described by Auffray & Rougeon [7]. The polyadenylated RNAs were selected by poly-U-Sepharose chromatography according to Sanchez et al. IS]. The RNA samples were electrophoresed on agarose gel by the formaldehyde procedure [4] and transferred to nitroceIlulose filters as reported by Thomas 191.

In-situ Hybridization Frozen tissue sections were prepared and processed as described by Hafen et al. [lo]. The probe was labelled by nick-translation with [3H]deoxynucleotides to 10’ cpm/ug DNA. The size of the fragments was monitored by a subsequent DNase I treatment (50 bp long) that allowed better penetration of the probe within the tissues. The hybridization was for 18 h in 50% formamide: 0.6 M NaCl, 10 mM Bis, pH 7.5,l mM EDTA, 1x Denhard’s mix, 100ug poly(A) at 38°C. These conditions correspond in situ to the conditions of high stringency on filters [ 111.The slides were then washed 24 h in the same buffer at 37°C and dipped in Kodak NTB2 emulsion. Exposure was for 3-4 weeks Exp Cell Res 163 (1986)

408 Le Parco et al.

Fig. 3. Localization of transcripts homologous to Dcgl in second-instar larvae. Both (A) brightfield and (B) dark-field photomicrographs of the same longitudinal section of a second instar larva are shown. The fat-bodies (FB) are the only structures displaying a strong and specific signal at this stage. (C, D) Photomicrographs represent a detail of the head region of another larva. PV, Proventriculus; B, brain. (A, B) x125; (C, D) x200.

at 4”C.The specificity of the signal was checked by hybridization on sections treated with RNase I, and by a parallel hybridization with the mid-gut-specific Jonah gene (clone aDM 3201 DH), a gift of Dr W. Gehring [lo] (not shown).

RESULTS AND DISCUSSION A Sau 3A partial digest genomic library of Drosophila melanogaster OreR strain in ;l-EMBL2 vector was screened for ‘collagen-like’ clones using the Pcg45 insert from the chicken pro-a2[I] cDNA clone [12]. Of the clones isolated Exp Cell Res 163 (1986)

Drosophila collagen gene expression 409

Fig. 4. Localization of transcripts homologous to Dcgl in third-instar larvae. (A) Bright-field and (B) dark-field photomicrographs of %&week exposure of a longitudinal section of the dorsal portion of the head (segments 4-6). The anterior lobes of the lymph glands are most heavily labelled on their periphery (LG), the fat bodies (FB), although highly refringent; the brain lobes (B) and the other structures are at the background level. (C, 0) Photomicrographs showing the hybridization signal detected over the ‘adepithehal cells’ associated with imaginal discs in the second half of the thirdlarva) stage. (0 Second; (0) third leg imaginal disc-epithelium Q are free of labelling, while ‘adepithelial cells’ (AE) lying in the lumen of the discs display a specific signal. These micrographs represent the most striking regions of the larva, but the other lymph-gland lobes and the adepithelial cells associated with the other imaginal discs are also labelled. (A, B) X 125, (C, 0) x200.

Exp Cell Res 163 (1986)

410 Le Pare0 et al. three were found to correspond to the Dcgl gene previously characterized by Monson et al. [3]. Indeed, restriction maps have shown similar patterns and especially those fragments cross-hybridizing with Pcg45 (fig. 1A, B). Moreover, their chromosomal location is the same (2L-25C, locus) and their nucleotidic sequences identical (results not shown). In addition to these results, the genomic hybridization patterns generated by these clones (fig. 1 C) lead to the conclusion that Dcgl is a single copy gene. The accumulation, during Drosophila developmental stages of the Dcglencoded transcripts was analysed by Northern blotting experiments. The results (fig. 2A) demonstrated that Dcgl probe hybridized to a 6.4 kb mRNA species, which is prominent during the first and second larval stages. However, it is noteworthy that this RNA species is also detectable, although to a lesser extent, during the embryonic stage, third instar larvae, white and old pupae and in adult flies. The conditions of hybridization in fig. 2 B (more labelled Dcgl probe and much more poly(A+) mRNA immobilized on the filters) even enabled us to detect a weaker expression in old pupae. Moreover, in addition to the 6.4 kb mRNA, a larger one was detected in these conditions during late embryonic and pupal stages. It is possible that this 10.0 kb species might represent a precursor for the major Dcgl transcript, as was suggested for other collagen mRNAs [13]. These results show that collagen-gene Dcgl is not stage-specific in its expression. To test whether Dcgl is differentially regulated during development in different tissues, we have performed in-situ hybridization experiments of the labelled Dcgl clone to frozen sections of successive developmental stages. We show here only the most striking figures (figs 3, 4); a more detailed study on the expression of this gene will be reported elsewhere. The results of in-situ hybridization are in agreement with those of Northern blots concerning the time of appearance of Dcgl transcripts. They demonstrate that even the weakest signals detected in fig. 2A actually correspond to striking concentrations of label. At the end of embryonic development, and during the first and second larval stages, the signal was specifically detected over the fat bodies (FB) (fig. 3A-D); which are formed during embryogenesis by cells arising from embryonic mesoderm [14]. This signal decreased rapidly during the course of third larval stage to drop to the background level in wandering larvae (fig. 4A, B). Meanwhile, a high and specific concentration of grains was detected, from the beginning of the third instar stage to up to the young pupa. It was localized over the ‘peripheral’ cells of the mesodermal lymph glands (fig. 4A, B). In parallel, several other clusters of grains were specifically observed during the second half of the third larval stage over groups of cells associated with the imaginal discs (fig. 4C, D). These cells have been called ‘adepithelial cells’, so as to identify them by their position in the discs [ 151, without implying a specific origin which is still a much debated Exp Cell Res I63 (1986)

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question [16-181. However, they were demonstrated to be mesodermal [19] and there is now no doubt that they are indeed one source of adult skeletal muscles [20, 211, but they are also thought to play other functions during disc development. This is not surprising, since, according to Lawrence & Brower [20] “these mesodermal cells do not appear as precisely determined, their development being, at least, partly dictated by the neighbouring ectodermal cells”. Lastly, specific intense signals were observed over mesodermal derivatives of the genital apparatus [22] of old pupae and adult flies (results not shown). The most significant conclusions brought by in situ hybridization are that (3 the expression of Dcgl collagen-gene is specific to a few related tissues, all of mesodermal origin, and that (ii) the accumulation of the transcripts is differential and stage-specific for each tissue. In addition, the data presented here give new important insights on Drosophila connective tissue production. These results give, for the first time, the evidence that Drosophila connective tissues are constituted, in part, by collagenous proteins. In cyclorrhaphan Diptera, the connective tissues display, in the form of basement membranes and ‘stroma’, an original continuity which suggests some community in origin and nature [23, 241. If the nature of these connective tissues in insects begins to be better described [21], their origin has not been clearly established. According to Ashhurst & Costin [25] “the paucity of critical developmental studies has led to conflicting opinions about the identity of the cells responsible for the synthesis of the collagenous tissues”. Indeed, according to Edwards et al. [26], all the cells should be capable of secreting extracellular matrix. But, the formation of basement membranes of some organs was thought to be carried out by the underlying epithelial cells [27]. In some tissues, Wigglesworth [28] considered that both the hemocytes, released in the hemolymph by the lymph glands and the underlying cells might play some part in connective tissue formation. The accumulation of Dcgl collagen-gene transcripts was localized particularly within the second instar fat bodies (FBs) and the third instar lymph glands, both mesodermal tissues possessing a connective tissue which not only ensheathes their lobes (basement membranes), but also extends between individual cells (stroma) [29, 301. This finding corroborates previous observations [30, 311, and should favour the hypothesis that the most likely source of connective tissue in an organ are the cells of this organ themselves. However, the demonstration that both the lymph-gland cells (hemocytes) and the cells associated with the third instar imaginal discs (adepithelial cells), actually synthetize the same specific type of collagen, tends to favour the hypothesis of El Shatoury 1181,that the lymph glands might be the source of the adepithelial cells. If this interpretation is correct, then our results should demonstrate that the wandering hemocytes, released by the lymph glands [32] do contribute in extracellular matrix composition in some tissues in the larva [16, 17, 251. Exp Cell Res 163 (1986)

412 Le Parco et al. The study we have undertaken on the Drosophila collagen-gene family would enable us to better define the exact nature, origin and specificity of all connective tissues in this organism. We thank Michele Cavalhn and Steve Kenidge for their instructive discussions and Serge Long for providing living embryos and larvae. This work was supported, in part, by an ATP CNRS no. 1138.

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