Characterization and mutant analysis of the Drosophila sema 5c gene

Authors


Abstract

Class V semaphorins are transmembrane glycoproteins characterised by the presence of thrombospondin type I (Tsp) repeats linked to their extracellular semaphorin domain. Sema 5C is the only class V semaphorin found in Drosophila. Dsema 5C RNA is maternally provided and its embryonic expression is prominent in the mesoderm and muscle attachment sites. Here, we show that DSema 5C exists in two protein isoforms as a result of alternative splicing and that both protein and RNA have similar expression patterns. Using a combination of various molecular markers, we show that the DSema 5C protein becomes enriched in mesodermal cells that would normally give rise to fat body and visceral structures. In late embryos, DSema 5C is expressed in segment boundary cells that would constitute subsets of muscle attachment sites. Both RNA and protein are excluded from the somatic precursors and the mature muscles. The expression data suggest DSema 5C localised to the epidermal component of muscle attachment sites. Mutations in Dsema 5C were isolated from a P-element excision screen and by blotting analysis. The Dsema 5C mutants are homozygous viable and show no obvious embryonic phenotypes, suggesting that the maternal and zygotic components of Dsema 5C are not essential for fly development. © 2001 Wiley-Liss, Inc.

INTRODUCTION

Semaphorins are a large family of transmembrane and secreted proteins that are conserved from insects to humans. Both vertebrates and invertebrates semaphorins have been shown to play important roles in axonogenesis with the potential of acting as chemorepellents/inhibitors or attractants (for reviews see Kolodkin and Ginty, 1997; Nakamura et al., 2000; Tamagnone and Comoglio, 2000). They are also shown to function in the development of non-neuronal tissues. Studies in mice have shown that mutations in class III semaphorins can cause defects in patterning and growth of not only nerves but also bones and heart (Behar et al., 1996). In addition, recent work in Caenorhabditis elegans has shown that Semaphorin-2a is required to prevent ectopic cell contacts during epidermal morphogenesis (Roy et al., 2000). The signals generated by semaphorins are transduced into cells by their receptors, plexins, and neuropilins (Kolodkin et al., 1997; Rohm et al., 2000; Tamagnone et al., 1999; Takahashi et al., 1999; Winberg et al., 1998). Interestingly, the semaphorin receptors, plexins, also contain semaphorin sequences in their extracellular domain (Winberg et al., 1998).

The semaphorin members, discovered in animals from diverse phyla, represent seven classes, all of which contain a large conserved semaphorin domain. The conserved semaphorin domain is ∼500aa in length. It contains 14–16 cysteines, many blocks of conserved residues, and no obvious repeats (Kolodkin et al., 1992, 1993; Luo et al., 1993). In most family members, the semaphorin domain is often linked to other extracellular sequence motifs, which are thought to influence its activity potential. Classes II, III, IV, and VII semaphorins also contain a single C2-type immunoglobulin (Ig) domain (Kolodkin et al., 1992). Class V semaphorins contain seven thrombospondin type 1 repeats (Tsp) but no Ig motifs (Adams et al., 1996). The Tsp repeats were originally identified in the extracellular matrix protein (ECM) thrombospondin (TSP-1; Adams and Lawler, 1993; Bork, 1993). They include the putative melanoma cell adhesive sequence, VTCG (Tolsma et al., 1993), and a novel heparin binding sequence, WSXW (Vogel et al., 1993), that mediates binding of certain cells to thrombospondin (for review see Adams and Tucker 2000).

Mutation analyses have been described for several members of semaphorin classes, namely I, II, and III, but not for class V semaphorins. However, the expression patterns of class V semaphorins suggest they may have more general biological roles. In the mouse, semF and semG are differentially expressed in embryonic and adult tissues. During early embryogenesis, they are expressed in complementary regions, with semF detected only in mesodermal cells, whereas semG expressed exclusively in the neuroepithelium. A possible role for class V semaphorins in axonogenesis was suggested from recent work on human Cris-du-chat syndrome. It was shown that class V semaphorin, semF, accounts for 10% of the region uncovered by the deletion, causing the neurological disorder (Simmons et al., 1998). In Drosophila, the RNA expression pattern of the only class V semaphorin gene, sema 5c, has recently been reported (Khare et al., 2000). sema 5C RNA is expressed in stage 10 oocyte and in early embryos, suggesting the presence of maternal contribution. During embryogenesis, the sema 5C RNA is also expressed in mesodermal tissues.

In this study, we characterize the protein expression pattern of the invertebrate class V semaphorin, Sema 5C, and report that mutants lacking Dsema 5C are homozygous viable with no detectable phenotypes.

RESULTS AND DISCUSSION

Isolation of cDNAs Encoding Alternatively Spliced Forms of the Dsema 5C Gene

Several cDNA clones encoding the Drosophila sema 5C gene were isolated during the analysis of ∼40 kb genomic DNA from the 68F cytological region (Fig. 1A). Dsema 5C contains 12 exons and occupies ∼14 kb of genomic DNA (Fig. 1A). The complete cDNA sequence of Dsema 5C has been previously reported by Khare et al. (2000). DSema 5C is an integral membrane protein of 1093aa (Figs. 1, 2), with a typical class V semaphorin structure consisting of a signal sequence at the N-terminus (aa1-28) followed by semaphorin domain (aa65-546), seven Tsps (aa554-948), transmembrane region (aa957-979), and cytoplasmic tail at the C-terminus. With the fly genome project concluded, Dsema 5C is the only class V semaphorin in Drosophila.

Figure 1.

Molecular organization of Dsema 5C. A: Schematic representation of Dsema 5C DNA and the structural features of its protein isoforms, DSema 5C and DSema 5C1. The genomic map of Dsema 5C region is shown on top. Downward arrow points to the position of EP(3)1049 insertion. The 12 exons of Dsema 5C are represented by rectangles. Horizontal arrows indicate the two putative in-frame translation start sites and the dark-filled circle indicates the stop codon. The extent of deleted genomic DNA in excision lines, E77 and E215, are represented by lines. E, EcoR1; S, Sal1. A polymorphic Sal1 site is shown in bold. The two protein isoforms, DSema 5C (1093aa) and DSema 5C1 (1029aa), are schematically represented below. SS, signal sequence (green); SEM, semaphorin domain (red); Tsp, Tsp repeats 1-7 (blue); TM, transmembrane domain (black). B: Western blot of total proteins isolated from control wt and Dsema 5C transheterozygous mutants of the genotype Dsema 5CE77/Df(3L)BK9 and Dsema 5CE215/Df(3L)BK9. The DSema 5C protein band detected in the control is indicated by arrow. Note the absence of this protein band from the Dsema 5C alleles. The numbers on the left represent the sizes of protein markers in KD. The lower panel (Tub) shows the same blot probed with anti-α-tubulin antibody as loading control; ITS is a 35S-Met labeled protein band obtained in in vitro translation experiment using Dsema 5C cDNA as template; the radioactive-labeled protein band is of similar size to that obtained in the Western blot (arrow).

Figure 2.

Amino acid sequence of the DSema 5C protein isoforms. A: Complete amino acid sequence of the DSema 5C protein isoform. The aa residues of Tsp repeats 2 and 3 that are removed in the DSema 5C1 isoform are shown in red. The N-terminal signal sequence is shown in bold. The semaphorin domain is underlined. Cysteine residues within the semaphorin domain are underlined and in bold. The 7 Tsps are bracketed. The putative transmembrane domain is italized and in bold. B: Alignment of the 7 Tsps of DSema 5C (T1-7) and the consensus sequence found in the Tsps of TSP-1 (Tc). The consensus sequence (Co) of the DSema 5C Tsp repeats contains residues that are present in three or more repeats. The identical CSVTCG motifs found in both Tsp repeat 6 of DSema 5C and TSP-1 are underlined. The conserved WSXW motif is shown in bold.

In addition to the full-length sema 5C cDNA reported by Khare et al. (2000), a second alternatively spliced version appears to exist. We named this alternatively spliced form Dsema 5C1. DSema 5C1 contains sequences for 6 Tsp repeats only (Figs. 1A, 2), as the splicing event in Dsema 5C reduces Tsp repeats 2 and 3 into just one repeat. Tsp type 1 repeats were originally found in TSP-1 and TSP-2 proteins, which are components of the extracellular matrix (Adams and Lawler, 1993; Bork, 1993). In the type 1 repeats of TSP-1, the CSVTCG and WSXW motifs have been identified as being essential for the heparin and cell binding properties (Guo et al., 1992; Li et al., 1993; Prater et al., 1991; Tolsma et al., 1993; Tuszynski et al., 1993; Vogel et al., 1993). The CSVTCG motif is completely conserved in Tsp repeat 6 and only partially conserved (CSXXCG) in the other Tsp repeats of DSema 5C (Fig. 2B). The WSXW motif is also completely conserved in Tsp repeats 1, 2, 6, and 7 of DSema 5C (Fig. 2B).

Dsema 5C RNA and Protein Are Expressed in Mesodermal Precursors and Muscle Attachment Sites

The Dsema 5C RNA is maternally deposited and its zygotic expression shows a dynamic pattern starting at early stages of embryonic development (Khare et al., 2000). At embryonic stages 7–9, the RNA is detected at relatively high levels in the ventral and dorsal regions of the embryo (Fig. 3A). Weaker lateral staining can also be seen giving the appearance of stripes. RNA staining is also detected in the gut primordia. At stages 9–11, the staining becomes intense mainly in 11 segmentally reiterated mesodermal patches and the gut primordia (Fig. 3B). Double labeling experiments with anti-Engrailed showed that Dsema 5C expressing cells are partially underneath and posterior to the epidermal Engrailed stripe at this stage (Fig. 3B). At embryonic stage 12, Dsema 5C expression is found in fat body cell clusters (Dunin-Borkowski et al., 1995; Riechmann et al., 1998), visceral structures, and in rows of cells at the segment borders, which later constitute attachment sites for subsets of muscles (Volk and VijayRaghavan 1994) (Fig. 3C). In late stage embryos, strong expression of Dsema 5C RNA is observed at muscle attachment sites but not detected in the muscles as can be seen in double labeling experiments with Dsema 5C RNA and anti-MHC antibody (Kiehart and Feghali, 1986) (Fig. 3D).

Figure 3.

Comparison of RNA and protein expression patterns. A–D and F–H show lateral views of embryos oriented anterior to the left and dorsal to the top. A–D and E–H are Dsema 5C transcript and protein expression patterns, respectively. A: High intensity staining in ventral patches, gut primordia, and the dorsal region of a stage 8 embryo; weak lateral staining can also be seen at this stage. B: RNA expression (blue) in a stage 11 embryo double labeled with anti-Engrailed antibody (brown); Dsema 5C RNA stains mesodermal cells located underneath and posterior to the Engrailed stripe. C: Stage 12 embryo showing staining in fat body clusters (arrowhead) and gut primordia; weak epithelial staining near segment borders can also be seen at this stage (arrow). D: Dissected stage 16 embryo double labeled for Dsema 5C RNA (blue) and anti-MHC antibody (brown) to reveal the muscle pattern; it shows RNA staining at attachment sites of segment border muscles (SBM, arrows) and ventrolateral muscles (VL); note the absence of RNA staining from the muscles. E: Ventral view of a stage 11 embryo showing protein expression in mesodermal cells stretching from tracheal pits dorsally (arrowheads in E and F) to the ventral midline (arrow). F: Lateral view of E. G: Stage 12 embryo showing protein staining in two fat body cell clusters per hemisegment (arrows). H: Dissected stage 16 embryo labeled with anti–DSema 5C antibody; it shows protein staining at attachment sites of SBMs (arrows) and VLs; note the absence of protein staining from the muscles. The specificity of the anti–DSema 5C antibody is validated by the absence of staining from control embryos homozygous for Df(3L)BK9 (J). An example of Dsema 5C overexpression with Gal4 drivers is shown in I; this early stage embryo shows RNA expression in the ventral furrow from a Dsema 5C transgene driven by twist-Gal4 driver.

Similar pattern of staining was also seen with anti-DSema 5C antibody (Fig. 3E–H). Like its RNA, the DSema 5C protein is also expressed in mesodermal precursor cells (Fig. 3E,F). At stage 11, the segmentally reiterated expression of DSema 5C becomes very prominent in a group of mesodermal cells that stretches from the tracheal pits at the dorsal side of the embryo to the ventral midline in each hemisegment (Fig. 3E,F). Similarly, at embryonic stage 12, DSema 5C expression is detected in fat body cell clusters (Figs. 3G) and double labeling experiments using anti–DSema 5C and fat body marker, anti-Serpent, showed coexpression of both proteins in these cell clusters (Fig. 4A–C). At late embryonic stages, anti–DSema 5C stains muscle attachment sites but are excluded from the muscles (Fig. 3H). Double labeling experiments with anti–DSema 5C, anti-bGal in the rp298 enhancer trap line (Nose et al., 1998), and anti-DMEF2 (Nguyen et al., 1994) muscle marker showed that DSema 5C is not present in muscle progenitors or the mature muscles (Fig. 4D–I). The specificity of anti–DSema 5C antibody was confirmed by double labeling of embryos obtained from the fly line, Df(3L)BK9/TM3Ubx-lacZ, with anti-bGal antibody. There was no anti–DSema 5C staining in embryos homozygous for the deletion (Fig. 3J). The expression data show that late in embryogenesis both RNA and protein are found at muscle attachment sites where each end of the muscle fiber is inserted and connected tightly to the specialized epidermal apodeme cell via the ECM. The absence of expression of both components from the muscles suggests DSema 5C localized on the epidermal surface of muscle attachments.

Figure 4.

Confocal images of DSema 5C expression in mesodermally derived tissues. A–I: Lateral views of embryos double labeled with anti-DSema 5C (green) and various molecular markers that label fat body (A, red), visceral and somatic precursors (D, red), and mature muscles (D, red). All embryos are oriented anterior to the left and dorsal to the top. C, F, and I are merged images of A and B, D and E, and G and H, respectively. A–I: Lateral views of embryos oriented anterior to the left and dorsal to the top. A–C: Stage 12 embryo double labeled with anti-Serpent antibody (red); anti-Serpent and anti–DSema 5C label the same fat body cell cluster (C, arrows). D–F: Double labeling with anti-bGal (red) and anti–DSema 5C (green) of a stage 12 embryo carrying the enhancer trap rp298, which is expressed in muscle progenitors; anti-bGal labels somatic (arrows) and visceral (arrowheads) precursors. DSema 5C staining is excluded from the somatic progenitors and only detected in intervening fat body cell clusters (F, arrows); partial overlap between anti-bGal and anti–DSema 5C stainings can be seen in the visceral region (F, arrowheads). G–I: Stage 16 embryo double labeled with anti-DMEF2 (red); anti-DMEF2 labels muscle nuclei whereas anti–DSema 5C is only present at muscle attachments and excluded from the muscles (I).

Isolation of Dsema 5C Mutants

Dsema 5C maps to 68F3 and several deficiencies that uncover this interval were obtained from Bloomigton stock center (see Experimental Procedures). In order to obtain specific alleles in Dsema 5C, we searched the Berkeley Genome Project Database for P-element insertions near the gene. In this search, EP(3)1049 was identified as a P-element insertion 82 bp upstream of the Dsema 5C gene (Fig. 1A). The EP-element was mobilized and 220 independent excisions were collected and analyzed on Southern blots. Two excision lines, E77 and E215, were found to delete into Dsema 5C. In E215, the genomic 1.6-kb Sal1 fragment containing exons 1, 2, and 3 is completely deleted whereas a part of the upstream adjacent 1.8-kb fragment is still present (Fig. 1A). E77 is a small deletion removing the Sal1 site located within the 5′ untranslated DNA sequence of the gene. In this mutant line, only a part of the 1.6-kb Sal1 fragment is deleted. Flies transheterozygous for these excision lines and a large deficiency, Df(3L)BK9, which removes the entire Dsema 5C gene, were also blotted and probed with the 1.6- and 1.8-kb Sal1 probes and similar results were obtained. We assume that Dsema 5CE215 is a null allele, because the mutants Dsema 5CE215/Df displayed no protein expression on Western blots (Fig. 1B). In addition, fly lines carrying the UAS-Dsema 5C transgene were generated and several Gal4 drivers were used for overexpression studies (Fig. 3I).

Dsema 5C Mutants Show No Obvious Embryonic Phenotype

The Dsema 5CE215 and Dsema 5CE77 mutant flies were viable either as homozygotes or as heterozygotes over Df(3)BK9. Homozygous stocks could be established. They did not show any readily detectable embryonic defects with the various mesodermal (anti-MHC and anti-DMEF2) and neuronal markers (22C10, BP102, anti-EVE, 1D4) used. Furthermore, overexpression experiments using various mesodermal, neuronal, and ubiquitous Gal4 drivers did not result in any obvious phenotypes. In conclusion, embryos lacking both maternal and zygotic Dsema 5C have no readily identifiable phenotypes. Double mutants carrying Dsema 5CE215 and mutations in the m-spo (Umemiya et al., 1997) and Dsema 1a (Yu et al., 1998) genes were also analysed. M-spo is an extracellular matrix protein, which, like DSema 5C, contains Tsp repeats and localises to muscle attachment sites. DSema 1a is a membrane-associated protein which, like DSema 5C, contains a large semaphorin domain and is found at muscle attachment sites. Null mutations in m-spo and Dsema 1a do not lead to any muscle attachment defects in the embryo and m-spo mutant homozygotes are viable and fully reproductive (Yu et al., 1998; Umemiya et al., 1997). The effects of the double mutant combinations on embryonic development and in particular on muscle attachment, since both proteins are localised to these sites, were studied. Double mutant embryos, lacking both maternal and zygotic m-spo and Dsema 5C, showed no muscle phenotypes. Similarly, the genetic combination with Dsema 1a did not result in any muscle attachment defects. In these double mutants, muscle attachments form normally.

The lack of muscle phenotype in Dsema 5C mutants is not surprising as muscle attachment sites are very complex structures rich in a variety of adhesion molecules. Mutations in several of these molecules result in mild or no muscle attachment phenotypes. For example, recent work describing Tiggrin, the PS2 integrin ligand, showed no abnormal muscle arrangement or structure in mutant embryos (Bunch et al., 1998). Muscle defects in tiggrin mutants were only detected in late 3rd instar larvae. In laminin A mutants, the PS1 integrin ligand, a majority of muscle fibers was inserted at their attachment sites normally, although defects were seen in the extension of some ventral muscle fibers (Henchcliffe et al., 1993). These and other studies (e.g., Fogerty et al., 1994; George et al., 1993; Saga et al., 1992; Yarnitzky and Volk, 1995) led to the suggestion that multiple components are involved in the stabilization of muscle attachments and that their overlapping functions are partially redundant.

The localization and structural features of DSema 5C are suggestive of roles for this protein in mediating cellular interactions and adhesive functions. The semaphorin domain in many semaphorin members has been shown to act as ligand for other receptors, namely plexins and neuropillins (for reviews see Nakamura et al., 2000; Tamagnone and Comoglio, 2000). The Tsp motifs have been shown in the extracellular matrix protein TSP-1 (Adams and Lawler, 1993; Bork, 1993) and other molecules to interact with a wide range of both matrix and cell surface receptors (for a recent review see Adams and Tucker, 2000). They have been shown to bind the cellular receptor CD36 (Asch et al., 1993) and integrin a3b1 (DeFreitas et al., 1995).

In conclusion, we have characterised Dsema 5C expression and generated mutations in this gene. The DSema 5C protein is expressed in fat body and visceral structures and localises to the epidermal apodemes of muscle attachments. The mutant analysis revealed no essential function for Dsema 5C in fly development.

EXPERIMENTAL PROCEDURES

Standard molecular biology techniques were carried out as described by Sambrook et al. (1989). Standard procedures involving Drosophila were performed as described by Ashburner (1989).

Mutagenesis

An EP line [EP(3)1049] located 5′ to the semaphorin gene was identified by sequence homology search. The EP fly insertion line was obtained from the fly genome project (Berkely). The EP element was mobilized using the immobile element P[ry+ Δ2-3](99B) as a transposase source (Robertson et al., 1988). Single jump starter males of the genotype EP1049/P[ry+ Δ2-3](99B) were crossed to 3rd chromosome balancer females. From each cross, one w-male was selected and crossed to 3rd chromosome balancer females. A total of 220 independent excision lines was established. These were analyzed on Southern blots using wt genomic DNA flanking the EP1049 insertion site as hybridization probes.

Fly Lines and Stocks

Two deficiencies uncovering the 68F interval, Df(3L)BK9 and Df(3L)vin7, were obtained from the Bloomington stock centre. The m-spoc26 null allele (Umemiya et al., 1997) and Dsema 1ap were used in generating double mutant stocks with Dsema 5C alleles. The Gal4 driver lines used for overexpression studies are twist-Gal4 for mesodermal driver, 24B for muscle driver, elav-Gal4 for neuronal driver, pGMR-Gal4 for eye driver, and heat shock and daughterless-Gal4 for ubiquitous drivers.

Isolation of Dsema 5C Genomic and cDNA Clones

Total genomic DNA was prepared from a fly line carrying a P-element insertion in the 68F region of the 3rd chromosome. Subcloning of the genomic DNA flanking the P-element was performed using standard plasmid-rescue techniques. The subcloned flanking DNA was used as a probe to screen an EMBL3 wt genomic library (Tamkun et al., 1992) and various cloned genomic fragments were subsequently used to screen plasmid (Brown and Kafatos, 1988) and lambda gt11 embryonic cDNA libraries. The cDNA clones were mapped with restriction enzymes and then used as probes in Northern hybridization and RNA in situ experiments.

Both strands of the cDNA clones were sequenced using universal M13 and gene-specific primers. The blast computer program was used to search the nonredundant GenBank, PDB, and SwissProt databases for sequence homology.

RNA In Situ and Immunostaining of Embryos

For RNA in situ, RNA probes were prepared using the Dig RNA labeling kit as described by the supplier (Boehringer Mannheim). Whole-mount Drosophila embryos were collected, fixed, and hybridized to the probe as described by Tear et al. (1996).

Anti-DSema 5C antibody was raised in mice using a 6His fusion protein as antigen. The fusion protein was generated in Escherichia coli by cloning a 0.6-kb Sal1/HindIII cDNA fragment carrying aa59-237 into PQR32 (QIAexpress vectors, QIAGEN). Following incubation of fixed embryos with the primary antibody and the horseradish peroxidase (HRP)-anti-mouse antibody, a modified horseradish peroxidase immunocytochemical method was used (Hsu et al., 1988). The specificity of the sera used was confirmed by showing a complete lack of staining in control animals carrying a deficiency that uncovers Dsema 5C [Df(3L)BK9]. Staging of embryos was according to Campos-Ortega and Hartenstein (1985). Photographs were taken using DIC optics with a Zeiss Axiophot microscope. For immunofluorescence, FITC- and Texas Red-conjugated anti-mouse or anti-rabbit antibodies were used. Confocal microscopy was done on a Biorad MRC 1024, using Adobe Photoshop for image processing.

Acknowledgements

We thank Kathy Mathews (Bloomington stock center) and Tod Lavorty (Fly Genome Project) for providing various fly lines, Mark Brennan (University of Louisville) for anti-Serpent antibody, Akinao Nose (Kyoto University) for providing us with reagents, HT Nguyen (Albert Einstein College of Medicine) for anti-DMEF2 antibody, and Sree Devi Menon (IMCB, Singapore) for her help in the project. We also thank Hing Fook Siong for his technical assistance.

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