DEVELOPMENTAL DYNAMICS 210:264?273 (1997) Novel Gene Containing Multiple Epidermal Growth Factor?Like Motifs Transiently Expressed in the Papillae of the Ascidian Tadpole Larvae JEREMY M. ARNOLD,1* RAJARAMAN ERI,1,2 BERNARD M. DEGNAN,3 AND MARTIN F. LAVIN1,2 Cancer Fund Research Unit, Queensland Institute of Medical Research, Brisbane, Australia 2University of Queensland, Department of Surgery, Royal Brisbane Hospital, Brisbane, Australia 3University of Queensland, Department of Zoology, Brisbane, Australia 1Queensland ABSTRACT We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis. We isolated 12 partial cDNA sequences which were expressed in a stagespecific manner using differential display. We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development. This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)?like repeats and three novel cysteine-rich repeats. Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo. As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis. Dev. Dyn. 1997;210:264?273. r 1997 Wiley-Liss, Inc. Key words: ascidian; EGF-like domain; gene expression; in situ hybridization; metamorphosis INTRODUCTION Ascidians are sessile marine invertebrates which develop rapidly into a motile tadpole larva with a simple body plan and small number of cells. Ascidian embryos are an important model for studies of cell determination. Specification of cell fate occurs early in development and is achieved mainly by the partitioning of cytoplasmic maternal factors into specific blastomeres. The cleavage pattern is invariant, and the complete cell lineage has been characterised up to the early gastrula stage (Conklin, 1905; Nishida, 1987; Nicol and Meinertzhagen, 1988). Interest in the ascidian model also stems from the fact that they are r 1997 WILEY-LISS, INC. primitive chordates, with a notochord and dorsal nerve cord evident at the larval stage (Berrill, 1955). The larva is a non-feeding dispersal phase which undergoes settlement and metamorphosis after a short period of swimming. The papillae secrete an adhesive material which enable the larva to settle, and following settlement, numerous morphogenetic movements and physiological changes transform the free-swimming larva into a fixed, filter-feeding juvenile (Satoh, 1994). Little is known about the identity of the signal for ascidian metamorphosis, although evidence suggests that it originates in the anterior part of the larval trunk near the papillae and has been suggested to be a diffusible molecule (Torrence and Cloney, 1983; Degnan et al., 1997). In order to investigate genes involved in ascidian development and metamorphosis, we have studied temporal changes in gene expression during the embryonic development of the tropical ascidian Herdmania momus (family Pyuridae) by mRNA differential display and northern blotting (Arnold et al., 1997). This resulted in the isolation of 12 partial cDNA sequences which were expressed in specific temporal patterns during development. Further characterisation of one of these clones resulted in the identification of a novel serine protease specifically expressed in the epidermis at the gastrula, neurula, and early tailbud stages (Arnold et al., 1997). We report here the isolation of the cDNA sequence for a gene which is specifically expressed during the late tailbud and larval stages of ascidian development. The predicted protein of 337 amino acids contains four epidermal growth factor (EGF)?like repeats, a motif of approximately 45 residues including six cysteines with highly conserved spacing which has been identified in over 70 different proteins, including many with diverse roles in growth and development (Davis, 1990; Campbell and Bork, 1993; Carpenter, 1993). Grant sponsor: Australian Research Council; Grant sponsor: Department of Surgery, University of Queensland. *Correspondence to: Jeremy M. Arnold, Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Road, Brisbane, 4029, Australia. E-mail: firstname.lastname@example.org Received 21 January 1997; Accepted 1 August 1997 GENE SIGNALS ASCIDIAN METAMORPHOSIS 265 Fig. 1. Temporal expression pattern of CB06LL18 as detected by differential display and northern blot analysis. A: Differential display pattern showing band CB06LL18. Duplicate PCR reactions are shown for maternal (M), gastrula (G), neurula (N), tailbud (T), and larval (L) cDNA. B: Northern blot analysis of CB06LL18. The filter containing 1 礸 of poly (A)1 RNA was probed against the partial cDNA fragment CB06LL18. Equal loading of RNA in all lanes was confirmed by ethidium bromide staining of the rRNA bands in the agarose gel and methylene blue staining of the rRNA bands on the filter after transfer. Analysis of the spatial expression pattern of this gene in tailbud and larval stage embryos by in situ hybridisation showed that its expression is localised throughout the papillae and most anterior end of the trunk. The similarity of this gene to other genes involved in signalling various events during development in other taxa and its highly restricted spatiotemporal expression pattern are discussed in relation to a possible role for this gene in signalling ascidian metamorphosis. amino acids. This predicted protein has a calculated molecular weight of 35.6 kDa and a very high proportion of cysteine (15%) and proline (12%) residues. This sequence has been deposited in Genbank with accession No. U82540. RESULTS Cloning and Characterisation of a Differential Display cDNA Product We have studied changes in gene expression during ascidian development using differential display to compare expression patterns in unfertilised eggs, gastrula, neurula, tailbud, and larval stages (Arnold et al., 1997). From this study, 12 partial cDNA sequences were cloned which were shown to be differentially expressed during development by northern blot analysis. In this study, the partial cDNA sequence designated CB06LL18 was selected for further characterisation. Northern blot analysis for this mRNA detects a single transcript of about 1.0 kb specifically expressed during the late tailbud and larval stages, with strongest expression at the larval stage (Arnold et al., 1997; Fig. 1). Nucleotide sequencing of the differential display partial cDNA sequence detected the sequence of the anchored primer T12MC at one end and the arbitrary 10mer at the other, suggesting that this clone may be located at the 38 end of the gene. A polymerase chain reaction (PCR) primer was designed for amplification of sequences at the predicted 58 end of this gene by rapid amplification of cDNA ends (RACE) (Fig. 2). This primer was used in combination with the adaptor primer (AP1), resulting in the amplification of a single PCR product of about 1100 bp from larval stage cDNA which was gel purified, cloned, and sequenced. PCR cycle sequencing was carried out on five independent clones, resulting in a continuous sequence of 1105 bp, of which 56 nucleotides overlapped with the 164-bp differential display clone CB06LL18; therefore the total sequence was 1213 bp (Fig. 2). Analysis of this sequence revealed a single major open reading frame of 1010 nucleotides from position 64 to 1074 coding for 337 Derived Amino Acid Sequence Analysis Searches of the EMBL database against the predicted amino acid sequence detected similarity with numerous other proteins over limited regions. The highest identity matches were to the human fibrillin 1 and 2 precursors, pig round worm chymotrypsin/elastase isoinhibitors 1 and 2?5, human von Willebrand factor precursor, mouse, rat, and human epidermal growth factor (EGF) precursors, mouse fibulin 1 and 2, Drosophila Delta and human TGF-b-binding proteins (Fig. 3). Analysis of repeat sequences within the ascidian protein in combination with the database searches demonstrated that the ascidian protein contains three types of repeat sequences. There are four EGF-like domains of 39 or 40 residues (defined by the presence of six cysteine residues with highly conserved spacing) located at positions 66 to 104, 162 to 200, 225 to 263, and 286 to 325, within which there is strong conservation of amino acid sequence (Fig. 3A). A second type of repeat sequence of eight residues follows directly on from three of these EGF-like repeats at positions 105 to 112, 201 to 208, and 326 to 333 (Fig. 3A). These EGF-like domains are most similar to non EGF-like sequences from the chymotrypsin/elastase isoinhibitor (ICE) proteins (1 and 2?5), and human von Willebrand factor precursor and to EGF-like domains from the mouse, rat, and human EGF precursors (EGF-like domain 2), and human fibrillin 1 and 2 (EGF-like domain 42) (Fig. 3A). The similarity with the chymotrypsin/elastase isoinhibitor proteins continues outside of the EGF-like domains to include a further six residues at each end. The third type of conserved region in the ascidian protein is a repeat of about 48 residues which also has six conserved cysteines, although the spacing of the fourth and fifth cysteine residues is divergent from the normal EGF-like consensus (Fig. 3B). There are two full repeats of this sequence located at positions 17 to 65 and 112 to 161 and a third fragmented repeat located at positions 210 to 224 and 261 to 285 (Fig. 3B). These domains are most similar to EGF-like domains from 266 ARNOLD ET AL. Fig. 2. Nucleotide and deduced amino acid sequences of gene CB06LL18: The upper line shows the nucleotide sequence while the lower line shows the derived amino acid sequence. Coding nucleotides are given in upper case, while non-coding sequences are given in lower case. The cDNA fragment corresponding to the differential display clone is given in bold type. The location of the oligonucleotide primer used for RACE is shown underlined. This sequence has been deposited in Genbank with accession number U82540. human fibrillin 1 and 2, Drosophila Delta, mouse fibulin 1 and 2, and human TGF-b masking protein; however, the regions of similarity span two tandemly repeated EGF-like motifs (Fig. 3B). We have called this gene HmEGFL-1 (Herdmaina momus EGF-like protein 1) in recognition of the presence of multiple EGF-like domains. The overall structure of the protein consists of three copies of a unit of about 96 residues consisting of one 48 residue repeat, followed by an EGF-like domain and an eight-residue repeat, although the third copy of this unit is interrupted by an additional EGF-like domain (EGF-like #3), as shown in Figure 4A. Analysis of the relative hydrophobicity of the ascidian protein using the Kyte-Doolittle scale (Kyte and Doolittle, 1982) with a window size of 7 (Fig. 4B) shows a highly hydrophobic region formed by approximately the first 20 residues, which is likely to represent a signal peptide. Analysis of this region for potential signal sequence cleavage sites using the von Hiejne method (von Hiejne, 1986) indicates that the most likely site for cleavage of a signal peptide is between Ala18 and Ile19. 267 Fig. 3. (Legend on page 268.) GENE SIGNALS ASCIDIAN METAMORPHOSIS 268 ARNOLD ET AL. Fig. 4. Domain structure of HmEGFL-1. A: The relative positions and approximate sizes of the predicted signal peptide, the 48-residue conserved sequence, the EGF-like domain, and the eight-residue sequence are shown. B: Hydrophilicity plot of HmEGFL-1 by the Kyte-Doolittle scale (Kyte and Doolittle, 1982). Since all homologies which were detected existed only in short regions within HmEGFL-1, we constructed a phylogenetic relationship based on the similarity of the EGF-like domains to sequences from 12 other proteins which were determined to have the greatest identity from database searches (Fig. 5). This analysis demonstrates that the ascidian EGF-like domains are not closely related to any of these sequences, but instead form a separate grouping. EGF-like repeat 3 is the most divergent of the four EGF-like domains and is grouped with the ICE proteins, although the assignment is tenuous due to the short branch length defining this group. the ascidian genome (Fig. 6). A major and a minor band were detected in both the BamHI (lane 1, 8 kb and 13 kb, respectively) and PstI (lane 4, 8 kb and 12 kb, respectively) digests. There were two bands of about equal intensity detected in the NcoI digest (lane 3, 12 kb and 16 kb) while a single band was detected in the HindIII digest (lane 2, 7 kb). This result suggests that HmEGFL-1 is present as a single copy per haploid genome in H. momus. The presence of a second, less intense band in the BamHI and PstI digests indicates that there is another related sequence in the genome. Determination of Gene Copy Number by Southern Blotting Temporal Expression of HmEGFL-1 During Development by Reverse Transcription (RT)-PCR Analysis Genomic Southern blotting was carried out to determine the number of copies of the HmEGFL-1 gene in To confirm the northern blot analysis of the temporal expression of HmEGFL-1 and extend it to include Fig. 3. Amino acid repeat elements within HmEGFL-1. A: Homology comparison between the 4 EGF-like domains and strongest matching regions from other proteins. The top four lines show the four EGF-like domains from the ascidian protein. The numbering on the left indicates their position within the protein. The six conserved cysteine residues are numbered. The bottom eight lines show the strongest matching sequences from a number of other proteins based on BLITZ searches against all four EGF-like domains from the ascidian protein. Gaps introduced for alignment of sequences are shown as spaces. The percent identity of all regions to the first EGF-like domain from the ascidian protein is given on the right. The proteins and the regions shown are chymotrypsin/ elastase isoinhibitor 1 (ICE 1), positions 17?61 (Babin et al., 1984); chymotrypsin/elastase isoinhibitors 2?5 (ICE 2), positions 16?60 (Babin et al., 1984); human fibrillin 1 (FBN 1), positions 2444?2484 (Pereira et al., 1993); human fibrillin 2 (FBN 2), positions 2494?2530 (Zhang et al., 1994); mouse epidermal growth factor precursor (EGF), positions 362? 402 (Scott et al., 1983; Gray et al., 1983); human EGF precursor, positions 356?396 (Bell et al., 1986); rat EGF precursor, positions 357?397 (Price et al., 1992); human von Willebrand factor precursor (VWF), positions 1153?1191 (Mancuso et al., 1989). B: Homology comparison of the three 48-residue conserved regions with the strongest matching regions from a number of other proteins based on BLITZ searches against the two complete motifs from the ascidian protein. The top four lines show the two complete 48-residue sequences from the ascidian protein and the two partial repeats. The six conserved cysteine residues are numbered. The next three lines show the alignment with human fibrillin 1 and 2 and Drosophila Delta. The last three lines show the alignment with mouse fibulin 1 and 2 and the human TGF-b?binding protein. The positions of the cysteine residues within the EGF-like domains are indicated. The proteins and the regions shown are human fibrillin 1 (FBN 1), positions 98?147 (Pereira et al., 1993); human fibrillin 2 (FBN 2), positions 128?166 (Zhang et al., 1994); Drosophila Delta (DELTA), positions 402?452 (Vaessin et al., 1987); mouse fibulin 1 (FBL 1), positions 283?329 (Pan et al., 1993a); mouse fibulin 2 (FBL 2), positions 820?866 (Pan et al., 1993b); human TGF-b?binding protein (TGFb BP), positions 685?728 (Kanzaki et al., 1990). GENE SIGNALS ASCIDIAN METAMORPHOSIS Fig. 5. Molecular phylogeny of the EGF-like domains from HmEGFL-1. The proteins and regions shown are as for Figure 3. Additional sequences are chick agrin, positions 1490?1522 (Tsim et al., 1992), bovine metallothionein III, positions 26?65 (Poutney et al., 1994), Plasmodium ookinete surface antigen precursor, positions 157?193 (Lal, 1990), and mouse thrombomodulin, positions 369?404 (Dittman and Majerus, 1989). The scale represents the number of differences between the proteins along the branches. post-larval stages, semi-quantitative RT-PCR analysis of neurula, mid-tailbud, larval, 48-hr post-metamorphosis, and adult gene expression was carried out (Fig. 7). This demonstrates that the strongest expression was at the larval stage. Weak expression was also detectable at the mid-tailbud stage, but only at longer exposures than shown in Figure 7. No expression was detected at the neurula stage, at 48 hr post-metamorphosis or in adults (Fig. 7). 269 Fig. 6. Genomic Southern blot analysis of the HmEGFL-1 gene. Genomic DNA (5 礸) was digested separately with BamHI (lane 1), HindIII (lane 2), NcoI (lane 3), and PstI (lane 4). The membrane was hybridised with random primed [32P]labelled probe and washed under high-stringency conditions. The numbers on the right show approximate size (kb). Analysis of the Spatial Expression Pattern of HmEGFL-1 To analyse the tissue specificity of expression of this gene at the tailbud and larval stages, whole-mount in situ hybridisation was carried out using sense and antisense digoxigenin-labelled riboprobes, whereby mRNA expression is detectable as purple staining (Fig. 8). The antisense riboprobe revealed staining restricted to the adhesive papillae and the most anterior part of the trunk in both late tailbud and larvae immediately after hatching (Fig. 8A,B). Staining was also detected in the larval tunic (tail fin) at the posterior end of the late tailbud and larval embryos; however, the same pattern was also detected with the sense control ribo- Fig. 7. RT-PCR analysis of the expression of HmEGFL-1. An autoradiograph of RT-PCR analysis of (A) HmEGFL-1 and (B) ubiquitin. The lanes shown are neurula (N), tailbud (T), larval (L), 48-hr postmetamorphosis (M), and adult (A) stages, respectively. probe (Fig. 8C), so this is attributed to non-specific trapping. No staining was detected in the papillae or 270 ARNOLD ET AL. Fig. 8. Spatial restriction of HmEGFL-1 expression by whole-mount in situ hybridisation. Expression of HmEGFL-1 is indicated by purple staining. Embryos were hybridised against antisense (A,B,D) and sense (C) riboprobes. A: A lateral view of a late tailbud embryo. Expression of HmEGFL-1 is observed at the anterior tip of the trunk. The posterior end of the tail is out of focus. B: A larval embryo immediately after hatching. Expression of HmEGFL-1 is observed in the papillae (pp) and anteriormost part of the trunk. The two dark spots are the ocellus (oc) and otolith (ot). C: A larval embryo hybridised with the sense riboprobe. No staining is detected in the papillae or anteriormost part of the trunk. The staining in the larval tunic at the posterior end of the tail is nonspecific. D: A saggital section of a larva after whole-mount in situ hybridisation. Strong expression is observed throughout the papillae (pp) and anteriormost part of the trunk. Weak expression is observed in the epidermis (ep). The larval tunic (tu) is nonspecifically stained. Anterior is to the right for all embryos. The Scale bar 5 100 祄 in A?C and 50 祄 in D. anterior part of the trunk with the sense probe (Fig. 8C). Expression above background was not detected in embryos before the late tailbud stage or in larvae 3 hr post-hatching. To analyse the cellular localisation of expression in more detail, some of the whole-mount antisense stained larvae were sectioned (Fig. 8D). This demonstrates that HmEGFL-1 is expressed strongly throughout all cells of the papillae and the most anterior part of the larval trunk. Less intense expression is also evident in the single layer of epidermal cells covering the entire trunk (Fig. 8D) of the larvae. DISCUSSION We have reported here the isolation and characterisation of the cDNA for a gene known to be specifically expressed during the tailbud and larval stages of ascidian development. The derived protein contains three types of repeat sequences, including four EGFlike repeats. The novel domain structure and lack of strong sequence homologies indicate that it represents a new gene which we have designated HmEGFL-1. The EGF-like domains had highest similarity to regions from the chymotrypsin/elastase isoinhibitor proteins (1 and 2?5), human fibrillin 1 and 2, the mouse, human, and rat epidermal growth factor (EGF) precursor, and human von Willebrand factor precursor. The similarity of the ascidian protein to the chymotrypsin/elastase isoinhibitor proteins extends a further six residues outside each end of the EGF-like domain. All eight cysteine residues in this region are conserved, although the spacing varies slightly. The chymotrypsin/ elastase isoinhibitor reactive site sequence of Leu, Met, Cys, and Arg (Babin et al., 1984) corresponds to a sequence of Leu, Ile, Cys, and Glu in HmEGFL-1, representing conservative changes to this sequence. The disulfide bonding structure of the chymotrypsin/ elastase isoinhibitor proteins is different from that of the EGF-like domains, and, as the degree of sequence similarity is not sufficient to determine which conformation is most likely for the ascidian protein, structural studies will be necessary. The homologous regions from human fibrillin 1 and 2 and the mouse, human, and rat epidermal growth factor (EGF) precursor are Ca21-binding EGF-like domains. These Ca21-binding EGF-like domains have, in addition to the six cysteine residues with conserved spacing, a secondary consensus of Asp/Asn, Asp/Asn, Asp/Asn, Tyr/Phe (positions 1, 3, 19, and 24 in Fig. 3A) (Rees et al., 1988; Davis, 1990). Studies have suggested that the role of Ca21 binding is to stabilise the conformation of the tandem EGF-like repeats which occur in these proteins (Rao et al., 1995; Knott et al., 1996). The ascidian protein does not contain this Ca21-binding consensus sequence which may reflect the fact that the EGF-like repeats in HmEGFL-1 are not in tandem, but are separated by other conserved regions. The 48-residue six-cysteine repeat does not have the conserved spacing of the fourth and fifth cysteines (CxC) found in EGF-like domains, although it may represent a divergent type of EGF-like domain as variations in the spacing of the fourth and fifth cysteine residues of up to four positions have been reported for fibulin-1 and fibulin-2 (Argraves et al., 1990; Pan et al., 1993b). The similarities of these 48 residue six-cysteine repeats were also against EGF-like domains, although in this case, the similarity spanned two tandemly repeated EGF-like domains. Structural studies of this motif will again be required to distinguish between the possibility of its being an EGF-like motif or another type of domain. The lack of overall sequence similarity to any known protein demonstrates that HmEGFL-1 represents a novel protein. The possibility of predicting function by sequence similarity is further complicated by the fact that EGF-like repeats are found in many proteins with GENE SIGNALS ASCIDIAN METAMORPHOSIS diverse roles in development such as cell growth and proliferation, specification of cell fate, and neuronal guidance (reviewed in Davis, 1990; Carpenter, 1993). The restricted spatiotemporal expression pattern of HmEGFL-1 indicates a role associated with the papillae and anterior part of the trunk at the larval stage of development. The papillae is a structure of packed cuboidal cells and some neuronal cells arranged in a triangular shape and covered with a single layer of flattened epidermal cells (Torrence and Cloney, 1983; Satoh, 1994). The papillae secrete adhesives which enable the larvae to attach to substrates, with attachment initiating the subsequent events of metamorphosis. Little is known about the identity of the signal for ascidian metamorphosis or how the various events are initiated or coordinated. A study of the role of the papillae in the metamorphosis of H. momus larvae has recently been reported (Degnan et al., 1997). In this study, the larvae were severed at various points along the anterior-posterior axis and the severed fragments were maintained in a common test. Anterior fragments could be induced to undergo metamorphosis; however, posterior fragments only underwent metamorphosis when fused with papillae-containing fragments (Degnan et al., 1997). These results suggest that an anterior signalling centre associated with the papillae releases a diffusible factor required for metamorphosis. The restriction of the spatial expression of HmEGFL-1 to the papillae and anteriormost trunk correlates with this demonstration of an anterior signalling centre of the larvae required for initiation of metamorphosis in H. momus (Degnan et al., 1997). Further, the predicted signal peptide in HmEGFL-1 and the lack of any other major hydrophobic regions which could form transmembrane domains suggests that HmEGFL-1 is likely to be an extracellular, secreted protein. This correlates with the demonstration that an intact nervous system is not required for induction of metamorphosis and that the signal can move posteriorly when cells are in direct or close contact (Degnan et al., 1997). Therefore, HmEGFL-1 may be involved in signalling the initiation of metamorphosis in H. momus. Molecular markers are now available for almost all cell types in the ascidian embryo. For the epidermis, six regions have been defined by restricted patterns of spatial gene expression (Ishida et al., 1996). HmEGFL-1 represents the first molecular marker described for the papillae and anteriormost trunk (region 3), and thus may be valuable for further studies of the differentiation of the papillae. EXPERIMENTAL PROCEDURES Ascidian Collection and Embryo Culture Adult specimens of H. momus forma curvata were collected from the slope and flats of Heron Reef (23�8 S; 151�8 E) and maintained in flow-though aquaria under constant illumination at The University of Queensland Heron Island Research Station. Embryos 271 were cultured into Millipore-filtered (0.2 祄 pore size) seawater (FSW) as described in Degnan et al. (1996). Metamorphosis was induced in larvae about 4 hr after hatching by adding KCl to 40 mM in the FSW (Degnan et al., 1997). RNA Isolation RNA for northern blots was isolated from large-scale cultures of stage-specific embryos (.5,000 embryos) obtained by the removal of unfertilised eggs from the mixed culture by centrifugation though Ficoll-Paque (Pharmacia) at 1,000g for 10 min; normal developing embryos were collected from the seawater?Ficoll interface. These embryos were of approximately 95% purity for the selected stage. RNA extraction was performed immediately by homogenising in 4 volumes of NETS buffer (50 mM Tris, pH 7.6, 200 mM NaCl, 50 mM EDTA, and 2% SDS), extracting with phenol/chloroform, and precipitating in ethanol. RNA was further purified by LiCl precipitation, and the concentration was determined by spectrophotometry. The extraction of poly(A)1 RNA was performed using an Oligotex mRNA kit (Qiagen) following the manufacturers instructions. Northern and Southern Blot Analysis Poly(A)1 RNA was resolved using formaldehydeagarose gel electrophoresis and transferred by capillary blotting to a Hybond-N membrane (Amersham) and fixed by UV irradiation (Sambrook et al., 1989). Membranes were prehybridised for at least 1 h at 42癈 in 50% formamide, 53 saline sodium citrate (SSC), 53 Denhardt?s solution, 50 mM sodium phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 250 礸/ml salmon sperm DNA, and 100 礸/ml yeast tRNA and hybridised for 16 hr in 50% formamide, 53 SSC, 13 Denhardt?s solution, 20 mM sodium phosphate buffer (pH 6.8), 1% SDS, 10% dextran sulfate, 100 礸/ml salmon sperm DNA, 100 礸/ml yeast tRNA, and approximately 100 ng of [32P]labelled DNA probe. Membranes were washed twice in 23 SSC/0.1% SDS at room temperature for 15 min and then once in 0.13 SSC/ 0.1% SDS at 65癈 for 15 min and exposed to film. Genomic DNA was extracted by lysing sperm in the NETS buffer containing 200 礸/ml proteinase K, followed by phenol/chloroform extraction and ethanol precipitation. This DNA was digested separately with BamHI, HindIII, NcoI, and PstI (5 礸 per digest), resolved on an agarose gel, and transferred to a nylon membrane by capillary blotting. The membrane was then hybridised with a [32P]labelled cDNA probe spanning positions 850 to 1105 of the HmEGFL-1 cDNA for 16 hr in a solution containing 53 SSC, 53 Denhardts solution, 5% dextran sulfate, 0.5% SDS, and 100 礸/ml salmon sperm DNA. Washing was carried out as above. RACE Cloning of cDNA The Clontech Marathony cDNA Amplification Kit was used according to the manufacturer?s instructions 272 ARNOLD ET AL. for the rapid amplification of cDNA ends (RACE) corresponding to clone CB06LL18. A PCR primer was designed to amplify the predicted 58 region of unknown sequence (58 GCTGCTTTTGAAGTCCCAACGC). Double-stranded cDNA was synthesised from 1 礸 of poly(A)1 RNA primed with an oligo-dT containing primer and ligated to the Marathon adaptor. PCR amplification was performed with the gene-specific primer and a primer that anneals to the adaptor: AP1 (58 CCATCCTAATACGACTCACTATAGGGC). The PCR product was gel purified before cloning into pGEM-5zf (Promega) and sequencing using the ABI Prismy Dye Terminator cycle sequencing kit. RT-PCR Analysis cDNA was synthesised from 1 礸 of stage-specific total RNA (DNase treated) using superscript reverse transcriptase (Gibco-BRL) and an oligo-dT primer in a 20 祃 reaction volume. PCR amplification was performed simultaneously for both HmEGFL-1 and ubiquitin using 1 and 2 祃 of a 1:100 dilution of this cDNA in a 25-祃 volume. Reaction conditions were 24 cycles of 94癈 for 40 sec, 58癈 for 40 sec, and 72癈 for 2 min. The primers used were EGF-1 (58 GATGGATCCGTCGGTGGAGCGATAGCC), EGF-2 (58 CAGGAATTCACTCGAATTCATCTGGGG), HmUBQ-1 (58 GAYAARGARGGNATHCCNCCNGAYCARCAG) and HmUBQ-2 (58 YTGDATYTTNGCYTTNACRTTYTCDAT). A control reaction without reverse transcription was included for each cDNA. The PCR products were resolved on duplicate agarose gels and transferred to nylon membranes by capillary blotting. These filters were then hybridized overnight to radioactive probes, one to HmEGFL-1 and the other to ubiquitin, washed, and exposed to film. To ensure consistency of results, the cDNA synthesis was carried out twice for each stage-specific RNA, and the PCR reactions were performed twice for each cDNA. The intensity of the bands was found to reflect the amount of input cDNA, and hence the amplification was quantitative and within the linear range of PCR amplification. Nucleotide and Amino Acid Sequence Analysis Nucleotide sequence analysis was performed using MacVector software. Database searches for derived amino acid sequence similarities were conducted at the National Centre for Biotechnology Information (NCBI) using the BLAST, BLITZ, and FASTA programs. For the construction of phylogenetic relationships, the Molecular Evolutionary Genetics Analysis (MEGA) software was used (Kumar et al., 1993). This software was used to construct a neighbour-joining tree (Satou and Nei, 1987) by the complete deletion method. Whole-Mount In Situ Hybridisation Whole-mount in situ hybridisation was performed using sense and antisense digoxigenin-labelled RNA probes as described (Arnold et al., 1997) based on a method originally described by Tautz and Pfeifle (1989). Briefly, embryos were fixed in 4% paraformaldehyde, washed in methanol, incubated in 90% methanol containing 50 mM EGTA, washed with 100% ethanol, and stored at 220癈 in 100% ethanol. After rehydration, the embryos were manually dechorionated in PBT (0.1% Tween 20 in PBS), treated with 50 礸/ml proteinase K for 15 min, and refixed in 4% paraformaldehyde in PBS. Prehybridisation was carried out in 50% formamide, 53 SSC, 0.1% Tween 20, 50 礸/ml heparin, and 100 礸/ml herring sperm DNA (HS) for 1 hr at 45癈 and hybridisation for 16 hr at 45癈 in a volume of 100 祃 containing 100 ng of digoxigenin (DIG)-labelled riboprobe. Riboprobes were constructed by the incorporation of digoxigenin (DIG)-UTP in an in vitro transcription reaction from the partial cDNA fragment CB06LL18 using either SP6 or T7 RNA polymerase to generate sense and antisense probes (Boehringer Mannheim DIG RNA labelling kit). Following hybridisation, the embryos were washed in 4:1, 3:2, 2:3, and 1:4 HS:PBT mixtures, then in PBT for 20 min each. The embryos were then treated with RNase A and RNase T1 and washed twice more with PBT. The anti-DIG antibody conjugate was preabsorbed against fixed embryos in PBT for 2 hr, and the embryos were incubated in PBT containing a 1:2,000 dilution of the antibody for 2 hr. Non-specifically bound antibody was removed by washing several times in PBT. The embryos were then washed twice in 100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2, and 0.1% Tween 20 and then incubated in the same buffer containing 4.5 祃 NBT/ml and 3.5 祃 BCIP/ml. 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