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Stage and tissue-specific expression of a collagen gene during Drosophila melanogaster development
Abstract
Based on data from developmental RNA profiles and in situ hybridization, we report a direct examination of the expression of one collagen gene (Dcg1) 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 Dcg1 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 larva.
... Diese Befunde stehen in Übereinstimmung mit der frühen Expression dieser Proteine (Wolfstetter et al., 2019). Während Perlecan wie Laminin-Untereinheiten und Nidogen bereits frühembryonal exprimiert wird (Friedrich et al., 2000), wird für die beiden Kollagen IV codierenden Gene, lediglich spätembryonale Expression im Fettkörper und in Hämozyten postuliert (Mirre et al., 1988;Monson et al., 1982;Natzle et al., 1982;Le Parco et al., 1986;Yasothornsrikul et al., 1997).Im Gegensatz zur voll ausdifferenzierten ECM ist die "frühe ECM" durch die Abwesenheit der Kollagen-Schicht und der unvollständigen Inkorporation von Perlecan, welches Kollagen für die korrekte Lokalisation benötigt (Matsubayashi et al., 2017;Pastor-Pareja and Xu, 2011), charakterisiert. Dabei liegt die Ursache in der fehlenden Sekretion von Kollagen durch Hämozyten. ...
... Die Expressionsanalyse der vorliegenden Arbeit zeigt starke mRNA Expression von Ndg in den mesodermalen Geweben, der viszeralen und somatischen Muskulatur sowie in den Kappenzellen (cap cells) der Chordotonalorgane. Es konnte allerdings keine Expression in embryonalen Hämozyten und im Fettkörper detektiert werden, die für die meisten ECM Proteine und im speziellen für Laminin und Kollagen IV die höchsten Expressionslevel aufweisen und für andere Gewebe sekretieren (Kusche-Gullberg et al., 1992;Martinek et al., 2008;Matsubayashi et al., 2017;Le Parco et al., 1986;Urbano et al., 2009;Wolfstetter and Holz, 2012 (Dahlitz, 2011). Im Gegensatz zu dem in dieser Arbeit durch in situ Hybridisierung sowie der weiteren durch Fluoreszenz in situ Hybridisierung detektierten Expressionsmuster (Wolfstetter et al., 2019) postulieren Dai et al. (2018) ...
... Some parts of the epithelium synthesize their own basement membrane proteins (Martin et al., 1999;Denef et al., 2008;Sorrosal et al., 2010). Meanwhile, hemocytes (blood cells) and fat body also synthesize basement membrane proteins, which are secreted to the hemolymph and deposited on various tissue surfaces (Le Parco et al., 1986; Kusche- (Prokop, 1999). During NMJ formation, RP3 motoneuron, which is a synaptic partner of muscles 6 and 7 (also known as VL3 and VL4), approaches the ventral side of the muscle 6/7 boundary and passes through the muscle 6/7 cleft (Chiba et al., 1993). ...
... Some parts of the epithelium synthesize their own basement membrane proteins, such as laminin and perlecan (Martin et al., 1999;Denef et al., 2008;Sorrosal et al., 2010). Meanwhile, hemocytes (blood cells) synthesize basement membrane proteins, such as Col IV and laminin, from embryonic stage (Le Parco et al., 1986;Kusche-Gullberg et al., 1992;Yasothornsrikul et al., 1997). During larval stages, Col IV is mainly produced by the fat body (Pastor-Pareja and Xu, 2011). ...
... Type IV Collagens are divided into two subfamilies, α1-like and α2-like, split already in Cnidaria (Aouacheria et al., 2006). Drosophila has two genes encoding α chains of Collagen IV, named viking (vkg) and Collagen at 25C (Cg25C) (Le Parco et al., 1986;Natzle et al., 1982;Rodriguez et al., 1996;Yasothornsrikul et al., 1997), belonging to the α2-like and α1-like subfamilies respectively. vkg and Cg25C are adjacently located head-to-head in the genome, an arrangement conserved in the three α1-like/α2-like pairs of Collagen IV genes in mammals. ...
... Once established a role for Viking in postembryonic development, we asked where the expression of Viking takes place during larval stages. In the embryo, even though Collagen IV is found in all tissues, mRNA in situ and enhancer traps suggest high expression in mesodermal derivatives (Le Parco et al., 1986;Rodriguez et al., 1996). In order to ascertain the source of Vkg protein, we made use of iGFPi in vkg G454 /+ larvae and knocked down expression of GFP-trapped Viking using Gal4 drivers with restricted expression patterns. ...
Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, we used a GFP-based RNAi method (iGFPi) designed to knock down any GFP-trapped protein in Drosophila. We found with this method that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. We also show that incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of Perlecan, which counters constriction by Collagen IV. Our results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form.
... It has been established that blood cells in Drosophila actively participate in the synthesis of extracellular matrix at all stages of development (review in Fessler et al., 1994). In situ hybridization experiments had shown (Le Parco et al., 1986) that the collagen Dcg1 gene is expressed in third instar larvae lymph glands. We used an antibody raised against peroxidasin that is specifically produced by macrophages at the embryonic stage for immunostaining. ...
... In larvae, the first lobes of the hematopoietic organ contain increasing numbers of differentiated blood cells, essentially secretory cells which are only found in the lymph gland. These first lobes produce proteins including collagen (Le Parco et al., 1986) and peroxidasin. In addition, after septic injury (P. ...
We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte.
... The BM is created through the release of its components from the fat body into the hemolymph and subsequent deposition onto the basal site of tissues, including the fat body itself. Hemocytes contribute to BM formation or its repair [32][33][34][35][36][37], although the relative contribution of hemocytes versus fat body may be variable [38]. In one study, the formation of BM deposits with similarity to fibrotic lesions was induced in the fat body by the plasma membrane overgrowth or alternatively through increased secretion of immune effectors. ...
Fibrotic lesions accompany several pathological conditions, including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components, including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apicobasal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide drosomycin (Drs), which also restores the secretory activity of the SGs. In addition to extracellular matrix components, both Drs and F-actin localize to fibrotic lesions.
... The BM is created through the release of its components from the fat body into the hemolymph and subsequent deposition onto the basal site of tissues including the fat body itself. Hemocytes contribute to BM formation or its repair [32][33][34][35][36][37] although the relative contribution of hemocytes versus fat body may be variable [38]. In one study, the formation of BM deposits with similarity to fibrotic lesions was induced in the fat body by plasma membrane overgrowth or alternatively through increased secretion of (which was not certified by peer review) is the author/funder. ...
Fibrotic lesions accompany several pathological conditions including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apico-basal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide Drosomycin (Drs), which also restores secretory activity of the SGs. In addition to ECM components, both Drs and F-actin localize to fibrotic lesions.
... The four main BM components are conserved in Drosophila (Table 1). A single variety of the collagen IV heterotrimer is formed by the products of Collagen at 25C (Cg25C, now officially named Col4a1) and viking (vkg), encoding α1and α2-like chains, respectively (Le Parco et al., 1986;Natzle et al., 1982;Pastor-Pareja and Xu, 2011;Yasothornsrikul et al., 1997). Two different laminin heterotrimers can form with alternative α chains encoded by genes Laminin A (LanA) and wing blister (wb), whereas unique β and γ chains are encoded by the Laminin B1 (LanB1) and Laminin B2 (LanB2) genes (Fessler et al., 1987;Kusche-Gullberg et al., 1992;Martin et al., 1999;Goodman, 1988, 1989). ...
The evolution of basement membranes (BMs) played an essential role in the organization of animal cells into tissues and diversification of body plans. The archetypal BM is a compact extracellular matrix polymer containing laminin, nidogen, collagen IV and perlecan (LNCP matrix) tightly packed into a homogenously thin planar layer. Contrasting this clear-cut morphological and compositional definition, there are numerous examples of LNCP matrices with unusual characteristics that deviate from this planar organization. Furthermore, BM components are found in non-planar matrices that are difficult to categorize as BMs at all. In this Review, I discuss examples of atypical BM organization. First, I highlight atypical BM structures in human tissues before describing the functional dissection of a plethora of BMs and BM-related structures in their tissue contexts in the fruit fly Drosophila melanogaster. To conclude, I summarize our incipient understanding of the mechanisms that provide morphological, compositional and functional diversity to BMs. It is becoming increasingly clear that atypical BMs are quite prevalent, and that even typical planar BMs harbor a lot of diversity that we do not yet comprehend.
... For example, the protein is found in the basement membranes around muscles, but it appears that it is other mesenchymal cells and not the muscle cells themselves that are depositing this protein. A high level of pro collagen IV transcript is associ ated with cells of mesodermal origin during the third ins tar larval stage, most notably with peripheral cells of the mesodermal lymph glands, adepi thelial cells of the imaginal discs, and mesodermal derivatives of the genital apparatus (Le Parco et al 1986b;Mirre et aI 1988). ...
... SPARC reduction led to a marked accumulation of BL components in the extracellular microenvironment of affected adipocytes. Temporal expression data from modENCODE (Graveley et al., 2011) indicate that maximum levels of SPARC and Col IV expression occur during the 1 st and 2 nd instar stages, with expression decreasing during the 3 rd larval instar prior to pupariation (Graveley et al., 2011;Natzle et al., 1982;Le Parco et al., 1986). Consistent with the idea that SPARC effects are largely mediated prior to the late 3 rd instar stage, knockdown of SPARC in 3 rd instar had no impact on survival or fat body remodeling (data not shown). ...
Background:
SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. We previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. We sought to explore if SPARC is required for proper BL assembly in the FB.
Results:
SPARC deficiency leads to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell-autonomous accumulation of BL components around mutant cells that include collagen IV (Col lV), Laminin, and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix.
Conclusions:
Our data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV.
... In other species, LH3 is expressed and its activity is required within collagen-producing cells where it facilitates the assembly and secretion of collagens – particularly type IV and type VI collagens. There are two type IV (but no type VI) collagen genes in Drosophila, Viking and Cg25C, which are expressed predominantly in haemocytes and fat body (Le Parco et al., 1986a,b; Yasothornsrikul et al., 1997). Collagen secreted by these cells contributes to the basement membrane of many tissues in the embryo. ...
The lysyl hydroxylase (LH) family of enzymes has important roles in the biosynthesis of collagen. In this paper we present the first description of Drosophila LH3 (dPlod), the only lysyl hydroxylase encoded in the fly genome. We have characterised in detail the developmental expression patterns of dPlod RNA and protein during embryogenesis. Consistent with its predicted function as a collagen-modifying enzyme, we find that dPlod is highly expressed in type-IV collagen-producing cells, particularly the haemocytes and fat body. Examination of dPlod subcellular localisation reveals that it is an endoplasmic reticulum resident protein, that partially overlaps with intracellular type-IV collagen. Furthermore, we show that dPlod is required for type-IV collagen secretion from haemocytes and fat body, and thus establish that LH3 enzyme function is conserved across widely separated animal phyla. Our findings, and the new tools we describe, establish the fly as an attractive model in which to study this important collagen biosynthesis enzyme.
... Although there is no proof that the Neb-colloostatin precursor is collagen, it is a possibility. At present, only one genelprotein sequence of preprocollagen a(1) IV of Drosophila has been published (Le Parco et al., 1986;Lunstrum et al., 1988;Blumberg et al., 1988). In this molecule, a (Gly-Xaa-Yaa), repeat is present over most of the length of the molecule with a few atypical regions. ...
During the purification of trypsin-modulating oostatic factor (TMOF) of the grey fleshfly Neobellieria bullata, a new factor with oostatic activity was discovered. We report herein its purification, primary structure and effects on oocyte development. Its amino acid sequence was determined as H-SIVPLGLPVPIGPIVVGPR-OH. Due to structural sequence similarities with parts of several known collagens and its oostatic activity, we named it Neb-colloostatin. The synthetic peptide inhibits yolk uptake by previtellogenic oocytes and might have a role in the absence of yolk deposition in penultimate oocytes. Neb-colloostatin does not inhibit trypsin biosynthesis in the gut or ecdysone biosynthesis by larval ring glands. It decreases vitellogenin concentrations in the hemolymph by an unknown mode of action. The role of extracellular matrix proteins in the feedback control of growth is discussed.
... Additionally, lamellocytes express an uncharacterized surface marker called L1 antigen (Asha et al., 2003). All Drosophila hemocytes specifically express the marker Hemese (He) (Kurucz et al., 2003), while a majority of plasmatocytes and crystal cells express the Collagens Viking and Cg25C (Le Parco et al., 1986;Yasothornsrikul et al., 1997) and the Von Willebrand-like factor Hemolectin (Hml) (Goto et al., 2003;Goto et al., 2001;Sinenko and Mathey-Prevot, 2004). ...
Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, we define the developmental steps in the maturation of blood cells (hemocytes) from their precursors. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis.
Basement membranes (BMs) are sheetlike extracellular matrices found at the basal surfaces of epithelial tissues. The structural and functional diversity of these matrices within the body endows them with the ability to affect multiple aspects of cell behavior and communication; for this reason, BMs are integral to many developmental processes. The power of Drosophila genetics, as applied to the BM, has yielded substantial insight into how these matrices influence development. Here, we explore three facets of BM biology to which Drosophila research has made particularly important contributions. First, we discuss how newly synthesized BM proteins are secreted to and assembled exclusively on basal epithelial surfaces. Next, we examine how regulation of the structural properties of the BM mechanically supports and guides tissue morphogenesis. Finally, we explore how BMs influence development through the modulation of several major signaling pathways.
Patched-related (Ptr) encodes a protein with 12 potential transmembrane domains and a sterol-sensing domain that is closely related in predicted topology and domain organization to Patched, the canonical receptor of the Hedgehog pathway. Here we describe the production of an antibody specific for Drosophila Ptr and analyze its spatial and temporal distribution in the embryo. We find that at early developmental stages Ptr is predominantly localized at cell periphery but later on it becomes strongly and almost exclusively expressed in hemocytes. Interestingly Ptr null mutant embryos died without hatching. Our findings suggest that Ptr plays an essential function in Drosophila development, perhaps as a new receptor of embryonic hemocytes.
We report the purification and characterization of three sequence-specific polyclonal antibodies raised against specific portions of the Drosophila αIV collagen chain produced from the gene DCg1. These antibodies were used for immunolocalization experiments on tissue sections from embryonic organogenesis stages (13–17) and first larval stages. This analysis was paralleled by in situ hybridization experiments with a labeled fragment of the gene DCg1. We demonstrated that, by late embryogenesis, the DCg1 αIV chain was synthesized by individual mesoblasts and deposited in basement membranes of skeletal and visceral muscles. These sites of αIV collagen deposition were the same, by first and second instars, but the protein was then synthesized by fat body cells. Our results were reminiscent of those obtained for vertebrate in vitro myogenesis, they suggested, moreover, a tissue-specific composition of basement membranes in Drosophila melanogaster.
Research on Drosophila extracellular matrix (ECM) has mostly concerned the pericellular matrix and its relation to cells during development. This chapter focuses on the preparation and properties of individual Drosophila ECM proteins. Homologies with the corresponding vertebrate proteins are notable for at least two major Drosophila ECM components, basement membrane collagen IV and laminin. They secrete ECM components into their culture media. The chapter describes how biochemical amounts of several ECM proteins are isolated from the conditioned media. Some of the progenitor cells from which these cell lines were derived may have been related to hemocytes or cells of the fat body. The relative electrophoretic mobilities of the nonreduced and reduced ECM proteins are also discussed in the chapter. The mobilities of ECM components may vary significantly from those of standard, commercial “marker proteins” for various reasons—the average mass residue of collagens is significantly less than that of marker proteins and highly charged proteins, such as glutactin, bind sodium dodecyl sulfate (SDS) abnormally.
We report a direct examination of the expression of one collagen gene (DCg1) during Drosophila melanogaster metamorphosis, based on data from in situ hybridization. The transcripts of this gene, thought to encode a basement membrane type IV collagen, are mainly accumulated during ecdysis in wandering haemocytes. Our results demonstrate that haemocytes contribute to extracellular matrix deposition and seem to perform a fibroblastic function during Drosophila development.
A collagen gene (Dcg1) was characterized in Drosophila melanogaster and shown to encode a peptide related to vertebrate basement membrane type IV collagen chains. To study the function of type IV collagen during Drosophila development, we transformed flies with a partially truncated Dcg1 gene under the control of a heat-shock promotor. This construct induced synthesis of shortened pro- chains which associated with normal ones and thereby caused degradation of the shortened and normal pro- chains through a process called pro-collagen suicide. A large proportion of embryos expressing the transgene developed a phenotype exhibiting absence or partial retraction of the germ band with defects in nerve cord condensation and dorsal closure. Together these results indicated that, during embryogenesis, type IV collagen was an essential guiding factor for cell-matrix interactions in morphogenetic events.
Details of the mechanisms that determine the shape and positioning of organs in the body cavity remain largely obscure. We show that stereotypic positioning of outgrowing Drosophila renal tubules depends on signaling in a subset of tubule cells and results from enhanced sensitivity to guidance signals by targeted matrix deposition. VEGF/PDGF ligands from the tubules attract hemocytes, which secrete components of the basement membrane to ensheath them. Collagen IV sensitizes tubule cells to localized BMP guidance cues. Signaling results in pathway activation in a subset of tubule cells that lead outgrowth through the body cavity. Failure of hemocyte migration, loss of collagen IV, or abrogation of BMP signaling results in tubule misrouting and defective organ shape and positioning. Such regulated interplay between cell-cell and cell-matrix interactions is likely to have wide relevance in organogenesis and congenital disease.
By means of immunocytochemistry with a battery of specific antibodies, we describe the expression of the collagen IV chain produced by the gene DCg1 during the two phases of Drosophila neurogenesis. DgC1 was not expressed in neuronal tissues as shown by in situ hybridization, but the onset of its expression in cells of mesodermal origin was concomitant with the appearance of collagen IV on early axon pathways and peripheral nerve roots in the embryonic developing CNS. A similar situation was found during imaginal CNS development at metamorphosis, when collagen IV immunoreactivity was detected on centrifugal and centripetal nerve pathways, and specially on retinula axons that develop from the eye imaginal disc towards the lamina anlage in the brain optic lobe. Our results strongly suggest that collagen IV could be involved, together with other informative molecules of basement membranes, in a dynamic process of cell-matrix interactions during the establishment of initial axon pathways and neurite outgrowth in vivo.
We have developed and characterized a battery of specific polyclonal antibodies directed against specific portions of the alpha-chain of collagen type IV synthesized in Drosophila by the gene DCg1. Here, we describe the use of these antibodies together with in situ hybridization experiments in an attempt to study the expression and localization of collagen type IV during Drosophila oogenesis and early embryogenesis. The results clearly demonstrate that DCg1 is maternally expressed by follicle cells and that the collagen type IV chain produced is stockpiled in the growing oocyte. During the gastrulation stages, this component of Drosophila basement membranes concentrated on cells involved in the gradual invaginations leading to morphogenetic furrows. The presence of collagen type IV, which is an RGD-bearing molecule, during early stages of Drosophila development is discussed in comparison with the crucial, active role its vertebrate counterpart is supposed to play in morphogenetic processes.
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The Pl gene, together with the LSP-1 alpha, -1 beta, and -1 gamma, LSP-2, and P6 genes, is expressed exclusively in the larval fat body of D. melanogaster during the third instar. In vivo mapping of the cis-acting regulatory sequences of the P1 gene was carried out using hybrid constructs with three different reporter genes and a combination of transient and germline transformation assays. This revealed that regulatory elements involved in the setting up of the temporal and spatial specificities of transcription of the P1 gene are located in a short DNA region immediately upstream of the mRNA transcription start. This region includes an element that behaves as a fat-body transcriptional enhancer and element(s) required for ecdysone inducibility of transcription of the P1 gene.
This chapter discusses the regulation of collagen protein synthesis of the cell, tissue, or whole animal level. Although in many cases these earlier studies pointed the way to the cellular and molecular biological experiments currently in progress. The collagens are a family of related proteins characterized by the repeating tripeptide sequence Gly-X-Y, in which X is often proline and Y is often hydroxyproline, and a triple-helical structure. This minimal definition of a collagen encompasses the twelve commonly recognized collagen types, a number of different collagens not yet accorded a type number, and several proteins whose structures and functions are sufficiently different from the collagens to warrant their exclusion from the collagen gene family. In addition, the effects of growth and differentiation factors on collagen expression during embryogenesis, tissue remodeling, wound repair, and normal morphogenesis are currently subjects of active study. Control of collagen synthesis in cells is complex and reflects both the translational and pre-translational mechanisms that are dependent on the cellular proliferative state. For most of the mediators discussed in this section, the net effect on collagen mRNA abundance has been shown, but the specific mechanisms remain to be established.
Type IV collagen, a major structural component of basement membrane, has been characterized only in vertebrates. It is unique among the collagenous proteins in that it forms specific lattice networks by end-to-end interactions. In particular, in mammals the C-terminal noncollagenous domain (NCl) of collagen IV was shown to be one of the major cross-linking sites in the network assembly. Here, we give the first direct evidence of type-IV-related collagen in invertebrates by sequence analysis of cDNA and genomic DNA clones for the 3'-end of a previously characterized Drosophila collagen gene. The data describe the C-terminal 190 amino acid residues of the triple helix and the entire noncollagenous domain (231 amino acids) of the chain encoded for by this gene. Comparison with data reported for human and mouse alpha 1(IV) chains reveals that triple-helix regions are quite different, while NC1 structures are very similar. This suggests different constraints on triple-helix and NC1 domains during evolution. Present data support the assumption that the NC1 structure originated from duplication of an ancestral sequence; the extent of both interspecies and intramolecular homologies suggests the maintenance in vertebrates and invertebrates of an ancestral specific function.
We have examined directly the expression of one collagen gene (DCg1) during Drosophila melanogaster embryogenesis by means of in situ hybridization. Transcripts of this gene, which were demonstrated to encode a basement membrane type IV collagen chain, began to accumulate specifically in mesodermal derivatives at stages 12-13 of embryogenesis, and not before. Cells expressing this gene overlap, or are closely intermingled with, somatic and visceral mesoderm in stages 12-14. In stages 15-17, in addition to the strongly positive fat bodies, highly labelled cell spots are found scattered around all the parts of the gut and symmetrically on each side of the ventral nerve cord. They correspond to circulating mesodermal cells which we consider to be haemocytes or mesoblasts.
A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.
We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.
The present study shows that hemocytic granular cells synthesize and secrete type IV collagen (ColIV) in the silkworm Bombyx mori (B. mori) and suggests that these cells play roles in the formation of basement membrane, the encapsulation of foreign bodies, and the metamorphic remodeling of the gut. The full- and partial-length cDNA of B. mori prolyl 4-hydroxylase alpha subunit (BmP4Halpha) and B. mori ColIV (BmColIV) were cloned, respectively. In situ hybridization and immunocytochemistry on larval tissues and cells identified hemocytic granular cells as the cells that express mRNAs and proteins of both BmP4Halpha and BmColIV. Immunohistochemistry and immunocytochemistry demonstrated that BmColIV was present in the basement membrane and in the secretory granules of granular cells, respectively. Granular cells in culture secreted BmColIV without accompanying the degranulation and discharged it from the granules when the cells were degranulated. Nylon threads were inserted into the hemocoel of larvae. Granular cells concentrated around the nylon threads and encapsulated them as a self-defense reaction. BmColIV was found to be a component of the capsules. Furthermore, the present study showed that actively BmColIV-expressing granular cells accumulated around the midgut epithelium and formed BmColIV-rich thick basal lamina-like structures there in larval to pupal metamorphosis.
(Fkh) is required to block salivary gland apoptosis, internalize salivary gland precursors, prevent expression of duct genes in secretory cells and maintain expression of CrebA, which is required for elevated secretory function. Here, we characterize two new Fkh-dependent genes: PH4alphaSG1 and PH4alphaSG2. We show through in vitro DNA-binding studies and in vivo expression assays that Fkh cooperates with the salivary gland-specific bHLH protein Sage to directly regulate expression of PH4alphaSG2, as well as sage itself, and to indirectly regulate expression of PH4alphaSG1. PH4alphaSG1 and PH4alphaSG2 encode alpha-subunits of resident ER enzymes that hydroxylate prolines in collagen and other secreted proteins. We demonstrate that salivary gland secretions are altered in embryos missing function of PH4alphaSG1 and PH4alphaSG2; secretory content is reduced and shows increased electron density by TEM. Interestingly, the altered secretory content results in regions of tube dilation and constriction, with intermittent tube closure. The regulation studies and phenotypic characterization of PH4alphaSG1 and PH4alphaSG2 link Fkh, which initiates tube formation, to the maintenance of an open and uniformly sized secretory tube.
The peripheral nerve branch innervating the femoral muscles of the common yellow jacket (Vespula carolina) has been found to possess a thick lemnoblast basement membrane and a complex mesaxon. The term "tunicated nerve" is proposed to designate the type of peripheral nerve in which one or several axons are loosely mantled by meandering, cytoplasm-enclosing membranes of the lemnoblast.
The peripheral axon courses longitudinally in a groove in the muscle fiber between the plasma membrane of the muscle fiber and a cap formed by lemnoblast and tracheoblast. The junction is characterized by apposition of plasma membranes of axon and muscle fiber, abundant mitochondria, and synaptic vesicles in the axon, and aggregates of "aposynaptic granules" plus mitochondria and endoplasmic reticulum on the muscle side of the synapse. Unlike the vertebrate striated muscle fiber, no complex infolding of the synapsing plasma membrane of the muscle fiber occurs.
The "connecting tissue" of the insect is formed by tracheoblasts, their basement membranes, and the basement membranes of other cells. Further mechanical support is given by the ramifying tracheoles.
The physiologic roles of the specialized structures are considered.
The embryological development of the connective tissue sheath around the nervous system has been investigated in Schistocerca gregaria. The sheath cells appear to be derived from outlying ganglion cells. The neural lamella is first visible when the embryo is 9 days old and it increases in thickness until hatching occurs on the twelfth day. It is produced entirely by the sheath cells.
The sheath cells have numerous lipid droplets in their cytoplasm. Some neutral mucopolysaccharide and proteins are also present. The histochemical reactions of the neural lamella after its formation suggest that it is composed of collagenous proteins embedded in neutral mucopolysaccharides.
The sheath cells are typical fibroblasts during the formation of the neural lamella. The cisternae of the endoplasmic reticulum are dilated into vesicles which contain a somewhat electron-dense material. No intracellular fibrils were observed. Collagen fibrils with banding of periodicity between 55 and 60 mµ. are seen in the neural lamella from 11 days onwards.
Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the latter two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.
A dot hybridization method is presented for rapidly determining the relative concentrations of nucleic acids in a mixture,
as well as the extent of sequence homology between related RNA or DNA species.
Asymmetric RNA probes, which contain only the mRNA coding strand, provide a large increase in hybridization efficiency in situ over that observed with either symmetric (both strands represented) RNA or DNA probes. Asymmetric RNA probes are synthesized in vitro by transcription from recombinants formed between sequences encoding sea urchin mRNAs and the transcription vector R7 delta 7. Using a probe representing early variant histone mRNA sequences we have characterized hybridization to sections of sea urchin embryos with respect to thermal stability of the hybrids formed, optimum temperature, effect of sequence divergence on hybrid thermal stability, and dependence of the hybridization signals on probe concentration and hybridization time. Estimates from the observed signals indicate that a large fraction of target RNAs is both retained in sections and hybridized with probe at saturation. Coupled with measurements of nonspecific background binding of heterologous probes, these data indicate that the method has sufficient sensitivity to detect many moderately abundant mRNAs (20-75 molecules per cell in the 1500-cell pluteus). In situ hybridizations to embryos at different developmental stages show that while histone mRNAs are uniformly distributed in cleaving embryos, different cell lineages of older embryos show large differences in accumulation of these mRNAs.
The amoebocytes, which form the most abundant type of blood-cell in Rhodnius, contain rounded, oval, or rod-shaped inclusions which stain by the periodic acid/Schiff method. These are believed to be neutral mucopolysaccharides. The amoebocytes apply themselves to the basal membranes, which likewise are PAS-positive, and appear to contribute to these membranes by discharging their contents. They insinuate themselves into the developing muscles and give rise to the sheaths of connective tissue by which the muscle fibres are surrounded. And they collect around deposits of injected Indian ink, producing similar sheets of PAS-positive material, presumably mucopolysaccharide.
The chitinous endocuticle is PAS-negative (except in a few special regions such as the neck and the conjunctival membranes of the limbs). It becomes positive during digestion by the moulting fluid. The tracheae react similarly.
Other PAS-positive structures which are not produced by the amoebocytes are the striated border of the mid-gut, the basement membrane of the gut, the perilemma around the ganglia and nerves, and the ground substance within the ganglia.
In insect embryos groups of epidermal cells are already determined1 to form particular compartments of the adult2-4. In Drosophila this determination is maintained through cell divisions, is stable to experimental interference1,5,6 and is thought to depend on the activity of a particular class of homoeotic genes4,7,8. In contrast to our understanding of the ectoderm, we know little about determination of the mesoderm, which gives rise to the musculature. A recent study of cell lineage in the thorax of adult Drosophila has shown that the muscles of each segment are divided precisely into sets (P.A.L., in preparation). The dorsal and ventral muscle sets of the mesothorax probably derive from adepithelial myoblasts9-11 in the wing and leg imaginal disk, respectively (P.A.L., in preparation). We describe here a new method of marking cells to trace myoblasts from the wing imaginal disk. We show that, when implanted, these mesodermal cells do not develop as they would in situ but instead contribute indiscriminately to developing host muscles. This result suggests that, whereas the ectodermal cells of the disk are committed to form a particular region of the adult, the mesodermal cells are not.
The fat body of Philosamia cynthia ricini grows gradually through larval life, the cells have the appearance of normal metabolizing cells except that the endoplasmic reticulum and Golgi elements are weakly developed. From the second instar there is a definite, layered, rather amorphous connective tissue sheath supporting the fat body. The sheath is largely destroyed during pupal histolysis. A new connective tissue sheath forms at the pupal-adult metamorphosis. During larval-pupal metamorphosis the ultrastructure of the fat-cells alters drastically, mitochondria become rare, the nucleus is distorted in shape, and a great variety of albuminoid grains appears in the cytoplasm. Glycogen grains are present in great quantity. The different types of albuminoid grains are described and classified into five main groups. It is argued that the grains are derived from mitochondria and other cell organelles by the action of lysosomes. Many of the grains are later destroyed during pupal histolysis either within the fat cells or after uptake by phagocytes. Most of the fat body is destroyed during this period, that which persists to the adult has a disordered cytoplasm, lack of glycogen granules, and bodies resembling degenerate mitochondria, which may be the result of a reversal of the process of grain formation. The nucleus appears degenerate at this stage.
1.Histological analyses were made of imaginal discs and histoblasts during the larval development ofDrosophila melanogaster to determine the number of cells, the patterns of cell division and the growth dynamics in these adult primordia. Histological studies were also made of the imaginal rings which are the primordia of the adult salivary gland, fore-and hindgut, the anlage cells of the midgut and several larval and embryonic tissues.2.In the newly-hatched larva, the immature eye-antenna, wing, haltere, leg and genital discs contain about 70, 38, 20, 36–45 and 64 cells respectively. These numbers include cells destined to form cuticular elements as well as peripodial, tracheal and nerve cells and probably the progenitors of adepithelial cells. The number of cells counted in the various imaginal disc anlagen is 1.5 to 4 times higher than the numbers deduced from genetic mosaic analyses by other investigators and reasons for these differences are given.3.About 12 h after fertilization, mitosis ceases in all tissues of the embryo except the nervous system. After the larva hatches, mitosis resumes in most of the imaginal anlagen and in some larval tissues. The time of resumption of mitosis in the imaginal anlagen was determined after treating the larvae with colchicine for 2 h.4.Among the imaginal discs, the eye disc is the first to begin cell division, at about 13–15 h after the hatching of the larva (first instar) followed by the wing (15–17 h), the haltere (18–20 h), the antenna, leg, and genitalia (24–26 h, early second instar), and finally the labial and dorsal prothoracic discs (52–54 h, early third instar). The cell doubling time for various discs was calculated from cell counts and the times agree closely with the doubling times deduced from clonal analyses by other workers: e.g., 7.5 h for the cells of the wing disc.5.The imaginal ring of the hindgut first shows cell division early in the second instar. The imaginal rings of the foregut and salivary glands, the anlage cells of the midgut and the cells of the segmental lateral tracheal branches begin to divide early in the third instar.6.The histoblasts which are the anlagen of the integument of the adult abdomen do not increase in number from the time of larval hatching until about 5 h after pupation when they begin to divide. Their behaviour contrasts with that of the histoblasts of the other dipterans such asCalliphora, Musca andDacus, which begin to divide during the second instar.7.The histoblasts are an integral part of the larval abdominal epidermis and, unlike imaginal disc cells, secrete cuticle during larval life. Each hemisegment consists of an anterior dorsal, a posterior dorsal, and a ventral histoblast nest containing about 13, 6 and 12 cells respectively. The 62 histoblasts in each larval segment represent about 7–8% of the total number of cells that form the integument of that segment.8.The number of cells in a particular type of histoblast nest was constant for both male and female larvae and among the different abdominal segments, except that the anterior dorsal group of the first and the seventh segments contains fewer cells than those of the other segments. Although the male and female adultDrosophila lack the first abdominal sternite and the male lacks the seventh abdominal tergite and sternite, the ventral histoblast nests of the first and the dorsal and ventral nests of the seventh abdominal segments are present in the larval stages as well as in the prepupa and have the same morphology and cell number as similar nests in the rest of the abdominal segments.9.The cells of the imaginal discs increase in volume about six-fold and their nuclei increase in volume three-fold between the time of hatching and the initiation of mitosis. The histoblasts increase in volume about 60-fold and their nuclei increase in volume about 25-fold between larval hatching and pupariation.10.Prior to each cell division, the nuclei of the columnar cells of the disc epithelium and of the histoblasts appear to migrate toward the apical surface of the epithelium. The cells round up and shift toward the apical region where mitosis occurs. After cytokinesis, the daughter cells move back to deeper positions in the epithelium. Because the nuclei of the non-dividing cells continue to lie deep in the epithelium, this intermitotic migration of nuclei gives these epithelia a pseudostratified appearance.11.Analyses of the growth of larval cells and of organs confirmed the observations of earlier investigators that cell division occurs only in a few larval tissues, whereas growth in the rest of the larval tissues is by cell enlargement and polyteny. During larval life, cell division was detected only in the central nervous system, gonads, prothoracic glands, lymph glands and haemocytes. Each tissue began mitosis at a characteristic stage in larval life. The larval cells that did not divide, grew enormously, e.g., epidermal cells increased in volume 150-fold and their nuclei increased in volume 80-fold.12.The adepithelial cells, which give rise to some of the imaginal muscles, were first identified between the thick side of the imaginal dise epithelium and the basement membrane at the beginning of the third larval instar (50–52 h). The origin of these precursors of mesodermal structures was analysed and evidence is presented that the adepithelial cells come from the disc epithelium. The question of the origin of the mesoderm of cyclorrhaphan Diptera is reviewed and it is suggested that the imaginal disc ectoderm may become segregated from the rest of the embryo before gastrulation has occurred, that is before the mesoderm has been established.
The ultrastructure of the imaginal discs ofDrosophila melanogaster was compared with that of other chitogenous tissues with different developmental capacities, namely, embryonic, larval, pupal and adult epidermis. Attention was paid to features which might be correlated with specific morphogenetic activities. Previous morphological studies of imaginal discs of Diptera were analyzed in detail and a somewhat revised view of imaginal disc structure emerged. The results reveal that the imaginal discs ofD. melanogaster consist of three types of cells: cells of the single layered disc epithelium, adepithelial cells and nerves. Four types of specialized junctions connect the cells of the disc epithelium: zonulae adhaerens, septate desmosomes, gap junctions and cytoplasmic bridges. The junctions are discussed in relation to their possible roles in adhesion and intercellular communication. It was concluded that gap junctions may be a more likely site for the intercellular communication involved in pattern formation than septate desmosomes. Evidence is presented that adepithelial cells are the precursors of imaginal muscles and that some cell lines (atelotypic) are in fact lines of adepithelial cells which can differentiate into muscle.
Specific imaginal discs can be easily recognized by their overall morphology, i.e. patterns of folds. However, no ultrastructural features were found which we could correlate with the state of determination of the cells. Most differences in the ultrastructure of different discs at several developmental stages were attributable to different phases of cuticle secretion. The cells of the imaginal disc epithelium are packed with ribosomes but very little rough ER. The amount of rough ER increases rapidly at puparium formation. Cuticulin is recognizable 4–6 hours after puparium formation. Six hours after puparium formation, the cells of the disc epithelium are secreting the epicuticle of the pupa. As the imaginal disc of a leg everts from a folded sac to the tubular pupal leg, the cells of the disc epithelium change from tall columnar to cuboidal. A loss of microtubules in the long axis of the columnar cells accompanies this change. Prepupal morphogenesis of the leg appears to be caused by the change in cell shape. Evidence is presented which is incompatible with previous explanations of the mechanism of eversion of imaginal discs.
There is some turnover of the cells of the disc epithelium as evidenced by autophagy and the occasional heterophagy of a dead neighbor. However this does not appear to be an important factor in the morphogenesis of discs. Plant peroxidase which was used as a tracer of proteins in the blood was taken up from the hemolymph by the disc epithelium. Imaginal disc cells contain many lipid droplets which coalesce and are replaced by glycogen during the prepupal period.
1.
A detailed morphological and histological description is given of the lymph glands of late third instar wild type larvae.
2.
In the late second instar of the wild type the lymph glands release some or their cells and these form the imaginal disc mesoderm, the lymph hearts, and other structures. The formation and the functioning of the lymph hearts are described.
3.
It is argued that blood cells do not exist in theDrosophila larva.
The structures formerly described as “blood-forming organs” or “lymph glands” in Drosophila melanogaster were re-examined by means of phase contrast, polarized light, and electron microscopy. As a result, the term “haemolymph organ” is suggested as more appropriate for these peculiar organs. Haemolymph organs of second and third instar larvae, and first stage (white) pupae from a wild strain (Varese) were studied; the methods are described in detail.
The haemolymph organ consists of two voluminous anterior lobes and several smaller posterior ones that are ovoid or tubular in shape. The anterior lobes comprise an outer capsule containing some muscle cells, a supporting stroma, and various kinds of cells. The stroma appears to consist of lamellated material of scleroprotein nature similar to that described in other organs of numerous insect species. This material must be considered as a component of the connective tissue characteristic of insect organs.
The cells are isolated or organized in groups of various sizes. The following types can be distinguished: rounded cells that are poorly differentiated, polygonal cells with well developed cytoplasm rich in organelles, polygonal cells with clear cytoplasm, and cells that appear to be intermediate form of the types mentioned above.
The morphological data presented confirm that the haemolymph organ produces and releases into the haemolymph various types of cells; of these the polygonal cells have a phagocytic and excretory function while only hypotheses can be presented at present concerning the role of the rounded cells. In addition the presence of polygonal cells with clear cytoplasm, showing signs of secretory activity, suggest that the haemolymph organ is involved in the production of substances constituting the fluid fraction of the haemolymph.
During the period between feeding and ecdysis in the fourth instar larva of Rhodnius the basement membrane below the epidermis of the abdomen increases about threefold in thickness. This increase takes place during the time, about 5 to 7 days after feeding, when the plasmatocytes settle and flatten in great numbers on the inner surface of the membrane.By a process beginning with pinocytosis the plasmatocytes accumulate their characteristic periodic acid Schiff (PAS)-positive inclusions, often with rod-like or tubular contents. These inclusions are discharged and spread out on the surface of the basement membrane. The inclusions and the membrane have the same staining properties and contain protein, carbohydrate, and lipid. Similar material is slowly deposited in less compact form between the plasmatocytes which encapsulate foreign bodies.The oenocytoids (granular cells) discharge the electron dense contents of their inclusions into the haemolymph rather later. The subsequent fate of this material is not known.
The ejaculatory duct of adult male locusts is surrounded by a thick layer of collagenous tissue. It starts to develop during the second instar when a group of cells come to lie adjacent to the basal regions of the epidermal cells which form the duct. From the fourth instar onwards, connective tissue is present between these cells. The matrix contains many collagen fibrils, some of which are very large and of irregular cross section. The banding periodicity is about 625 A. The cells differentiate into typical fibroblasts with highly dilated rough endoplasmic reticulum and small Golgi complexes. In the sexually mature adult, the cells are smaller and appear less active.
Recombinant bacterial plasmids have been constructed by inserting double-stranded chicken procollagen cDNA sequences linked to chemically synthesized decanucleotides containing HindIII sites into the HindIII site of pBR322. After transformation of Escherichia coli chi1776, colonies were selected by ampicillin resistance and recombinants containing procollagen sequences were identified by colony hybridization to 32P-labeled procollagen cDNA. The inserts from three recombinant plasmids, pCg10, pCg13, and pCg45, were 1200, 2200, and 2550 base pairs long respectively. Their sequence homology has been established by restriction mapping and crosshybridization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts, pCg45 has been positively identified as containing the pro alpha2 collagen sequence by partial determination of the DNA sequence of its ends: it has a short thymine-rich sequence at one end and a sequence coding for residues 478--499 in the chicken alpha2 chain at the other end.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
By making genetic mosaics and constructing embryonic ``fate maps'' it is
possible to locate the anatomical site of abnormalities affecting
behaviour.
A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently.
We report the structure and developmental expression of collagen gene sequences in Drosophila melanogaster. Collagen-like genomic clones were isolated by screening a Drosophila genomic library with a chicken pro alpha 2(I) cDNA clone as a hybridization probe. A 1.5-kilobase (kb) DNA sequence from a 9.2-kb DNA clone (pDCg1) is presented. Unlike the highly fragmented genes for vertebrate type I collagen, there is no evidence of a 54-base-pair primordial unit within this gene segment. Instead, the fragment is composed of two large coding sequences. Together they specify a sequence of 469 amino acids. This collagen product is composed almost entirely of the Gly-X-Y repeat characteristic of peptides involved in triple helix formation. Within the polypeptide there are four minor discontinuities in the Gly-X-Y pattern. Similar interruptions have been observed in a mouse basement membrane collagen protein sequence. Therefore, the Drosophila collagen gene may encode a nonfibrous collagen such as a basement membrane or cuticle collagen or a novel collagenous protein. By using the DNA segment of known sequence as a hybridization probe, a developmental sequence of polyadenylylated RNA samples was screened for the presence of homologous sequences. A RNA species 6.4 kb in length was detected as a prominent band only in the first- and second-instar larval stages. This pattern of developmental hybridization correlates with the production of the cuticle and basement membranes, and the large size of the RNA is consistent with its identification as a collagen-encoding RNA.
A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.
Much of the present interest in vertebrate collagens stems from the important part which these extracellular, structural proteins play in developmental processes and tissue organization as well as from their complex gene structure. So far the only vertebrate collagen genes examined encode the constituent polypeptide (pro alpha) chains of type I procollagen, that is, the pro alpha2(I) genes from chicken1,2 and sheep3, and the pro alpha1(I) gene from mouse4. Recently, we have isolated several collagen-like genomic DNA clones from Drosophila melanogaster5. In addition to providing data on the evolutionary history of this gene family, studying Drosophila has distinct advantages for cytogenetic localization of genes and for defining the functional roles of individual collagens by the application of genetic techniques. Here we compare the hybridization patterns, cytogenetic localization and expression of two of the Drosophila clones, DCg1 and DCg2. Although they are cytogenetically unlinked, they share similar developmental RNA profiles.
The thorax of the adult Drosophila contains about 80 muscles, which develop from the mesoderm. A new genetic marker was used to map the cell lineage of the myoblasts that form these muscles. Clones of marked cells were produced by irradiation of embryos and larvae, and these were detected in the adult by histochemical staining. The principal findings are that the muscles of each segment have separate origins, and that each becomes compartmented precisely into a dorsal-lineage and a ventral-lineage set of muscles, each set probably being formed by the adepithelial cells found in one imaginal disc. In contrast with the epidermis, the muscles of each thoracic segment are not subdivided into anterior and posterior compartments, and clones of muscle cells that are homozygous for recessive-lethal alleles of engrailed develop normally.
The peripheral nerve branch innervating the femoral muscles of the common yellow jacket (Vespula carolina) has been found to possess a thick lemnoblast basement membrane and a complex mesaxon. The term "tunicated nerve" is proposed to designate the type of peripheral nerve in which one or several axons are loosely mantled by meandering, cytoplasm-enclosing membranes of the lemnoblast. The peripheral axon courses longitudinally in a groove in the muscle fiber between the plasma membrane of the muscle fiber and a cap formed by lemnoblast and tracheoblast. The junction is characterized by apposition of plasma membranes of axon and muscle fiber, abundant mitochondria, and synaptic vesicles in the axon, and aggregates of "aposynaptic granules" plus mitochondria and endoplasmic reticulum on the muscle side of the synapse. Unlike the vertebrate striated muscle fiber, no complex infolding of the synapsing plasma membrane of the muscle fiber occurs. The "connecting tissue" of the insect is formed by tracheoblasts, their basement membranes, and the basement membranes of other cells. Further mechanical support is given by the ramifying tracheoles. The physiologic roles of the specialized structures are considered.
An improved method for the detection of cellular RNAs in tissue sections has been developed. It involves in situ hybridization of tritium-labeled cloned DNA probes to tissue sections and autoradiography. The method was calibrated by using a cloned DNA probe complementary to transcripts abundant in the midgut cells of Drosophila larvae. The improved method also permitted the detection of these transcripts in sectioned embryos where they are much less abundant. The sensitivity of the method can be approximated by quantifying the signal intensities over the hybridizing embryonic midgut cells relative to the larval midgut cells for which the number of transcripts has been estimated. Based on these calculations we estimate that the method is sensitive enough to detect 100 complementary RNA molecules per cell after 3 days of autoradiographic exposure with a signal-to-noise ratio of 10. The method has been successfully applied to detect transcripts of the homeotic gene Antennapedia. Serial sections allow us to study the spatial pattern of gene expression in the course of development.