MOLECULAR REPRODUCTION AND DEVELOPMENT 29:313-322 (1991)

Structure and Expression of the Nerve Growth Factor Gene in Xinopus Oocytes and Embryos FILOMENA CARRIERO, NADIA CAMPIONI, BEATRICE CARDINALI, PAOLA PIERANDREI-AMALDI Istituto di Biologia Cellulare, C.N.R., Rome, Italy A large part of the coding portion ABSTRACT of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out. Key Words: NGF, Development, Xenopus laevis

INTRODUCTION During development of the vertebrate nervous system the nerve growth factor (NGF) plays an important role in the survival and maintenance of the sympathetic and sensory neurones and has a differentiative effect on several populations in the central nervous system. Injection of purified NGF into developing animals elicits growth and differentiation of these cells, whereas administration of antibodies against NGF induces them to die (Levi-Montalcini and Booker, 1960). NGF is the first growth factor that has been shown to be responsible for neural survival in vivo and whose activity has been extensively studied (for a review, see Levi-Montalcini, 1987). More recently other factors related to NGF have been discovered which also carry out a neurotrophic function, suggesting that they are members of a gene family (Barde et al., 1982; Hohn et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., o 1991 WrmY-LIss, INC.

AND

1990). It has been shown that responsive cells have NGF receptors on their cell bodies and on their processes, and that they acquire NGF by a retrograde axonal transport system (Johnson et al., 1978; Herrup and Thoenen, 1979). The multiple morphological, chemical, and molecular changes elicited by NGF administration imply a complex mechanism of action (Yanker and Shooter, 1982; Levi et al., 1988). Several studies have been carried out on the structure and expression of the gene for NGF in some mammals and in the chicken. These have suggested a correlation between the expression of NGF by a target tissue during neural development and the density of sympathetic innervation in peripheral organs (Korshing and Thoenen, 1983; Shelton and Reichardt, 1984; Goedert, 1986; Ebendal and Persson, 1988). They have also indicated that the expression of NGF by a particular target tissue selectively influences the type of innervation (Edwards et al., 1989);however, in sensory deprivation experiments the level of NGF transcripts appears to be independent of the formation of certain neuronal connections (Selby et al., 1987b). Interestingly, in the mouse system it was found that transcription of the NGF gene results in four different mRNA species, which can be accounted for by independent initiation from two promoters and by alternative splicing (Edwards et al., 1986; Selby et al., 1987b). Moreover, transfection experiments in a variety of cell types have shown that the proteins encoded for by two of the different transcripts are processed by an identical pathway, yielding a common NGF precursor (Edwards et al., 1988). Most studies on NGF have been performed in high vertebrates, whereas little attention has been paid to low vertebrates. Among these Xenopus laeuis offers several advantages for developmental studies. A report on the effect of injected murine NGF into premetamorphic Xenopus tadpoles has shown that this factor enhances differentiative processes of sensory and sympathetic cells and also determines growth and differentiation of neurons in the central nervous system (Levi-Montalcini and Aloe, 1985). With the present study we start enquiring about a possible role of NGF in development of the nervous system during early ~

Received February 4, 1991; accepted March 25, 1991. Address reprint requests to P. Pierandrei-Amaldi, Istituto di Biologia Cellulare C.N.R., via Marx 43, 00137 Rome, Italy.

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RNA Preparation and Analysis For RNA preparation, groups of 1,000 oocytes or 300 embryos of different stages were frozen at - 70°C. They were then homogenized in Proteinase K/SDS solution and treated with phenolichloroform to extract nucleic acids as previously described (Pierandrei-Amaldiet al., 1988). When preparing polysomes, 400 staged oocytes or embryos (not frozen) were homogenized in the presence of 300 Uiml of RNase inhibitor, nuclei were pelleted at 2,000 rpm for 5 minutes, and the cytoplasmic extracts were fractionated on 1 5 4 0 % sucrose gradients as previously described; extract corresponding to no more than 100 individuals was loaded on each MATERIALS AND METHODS gradient (Pierandrei-Amaldi et al., 1985).The fractions Biological Materials corresponding to polysomes and mRNP were ethanol precipitated and the RNA was extracted as described Xenopus laeuis adults were purchased from Holmsdale Nursery, Redhill, UK and maintained in running above. Poly(A) + RNA was selected by oligo-dT celluwater. Ovaries were disaggregated with collagenase lose columns, electrophoresed in agarose gels containand the oocytes prepared as previously described ing formaldehyde, and transferred to nitrocellulose (Pierandrei-Amaldi et al., 1982); oocytes were staged filters. Each lane was loaded with the RNA equivalent according to Dumont (Dumont, 1972). Embryos were to a fixed number of oocytes or embryos. The filters obtained by artificial fertilization as described (Pieran- were hybridized to homologous probes, labeled with drei-Amaldi et al., 1988)and staged according to Nieuw- 32P,in 50% formamide at 42"C, washed in 0.1 x SSC at 52"C, and exposed to X-ray films with an intensifying koop and Faber (1956). screen. Construction and Screening of a Partial In Vitro Transcription and Translation Genomic Library A NsiI-AccI fragment of the third exon of the gene Xenopus laeuis DNA (100 pg) extracted from erythrocytes of a single frog was digested with EcoRI and spanning from nucleotide 125 to nucleotide 1039 was separated on a preparative 0.8% agarose gel. The cloned into the transcription vector pSP65AT and after region of the gel containing fragments of 7.4 kb was linearization at the appropriate restriction site, synexcised and electroeluted. Sixty nanograms of these thetic capped and polyadenylated mRNAs were generfragments was ligated with 1 pg of the lambda g t l l ated and prepared for in vitro translation (Krieg and DNA digested with EcoRI, packaged, and used to infect Melton, 1984). Synthetic mRNA was translated in Escherichia coli Y1088 cells. Plaques were screened by rabbit reticolocyte lysates as described by the manuhybridization to a chicken NGF probe, prepared by the facturer (New England Nuclear). random primed method, in 50% formamide at 37°C and Protein Analysis washed in 2 x SSC at 45°C; these conditions were Protein extracts were prepared from defolliculated always used for hybridization with the heterologous probe. Phages from positive plaques were grown, and oocytes and dejelled embryos (Pierandrei-Amaldi et al., the DNA was digested with EcoRI and subjected to 1982) by homogenization in 10 mM Tris-HC1 pH 6.8, Southern blotting. The insert was subcloned in the and spinning the postnuclear supernatant at 33,000 EcoRI site of the vector pSP65. Cloning procedure was rpm for 1hour in the ultracentrifuge. The supernatant carried out according to standard techniques (Maniatis was dialyzed overnight at 4°C against distilled water, clarified by spinning for 15 minutes in the microfuge, et al., 1982). and frozen at -70°C in aliquots. Restriction and Sequence Analysis The samples were loaded on 12% polyacrylamide A AccI-Hind111subfragment of 2.3 kb, still positive to SDS gels and analyzed by western blotting according to hybridization, was subcloned in the corresponding sites standard procedures (Towbin et al., 1979). The affinity of the vector pSP65. This fragment was subjected to purified rabbit antibodies against mouse-NGF were sequence analysis following the method of Maxam and used at a concentration of 10 pgiml. Antibody binding Gilbert (1980). Sequence was determined on DNA was visualized with an alkaline phosphatase kit-BCIPi NBT color development (BioRad). digested with HinfI, with DdeI, or with HinfI-RsaI. The 2.3 kb fragment digested with HinfI was blotted RESULTS and hybridized to the chicken probe: only a 328 bp Identification and Characterization of the fragment gave a high signal. Preparation of this fragXenopus NGF Gene ment was performed by separation in low melting Preliminary experiments aimed to identify the NGF agarose; the band was directly used for making a probe sequence in a tadpole cDNA bank failed, probably due by the random primed method. embryogenesis. In fact the importance of diffusible substances, related to growth factor molecules, in inducing cell differentiation is becoming progressively evident in Xenopus development (for reviews, see Gurdon et al., 1989; Dawid et al., 1990). With this in mind we have cloned and characterized most of the coding portion of the Xenopus NGF gene and compared it with that of NGF of other vertebrates. We have also analyzed the pattern of expression of this gene during oogenesis and embryogenesis by measuring the accumulation of NGF mRNA at different developmental stages.

XENOPUS NGF GENE to the low abundance of its mRNA; thus we decided to construct a partial genomic library from the DNA fragments that hybridized, although weakly, to a heterologous probe. In fact using a chicken NGF cDNA clone (Goedert, 1986) as a probe, it was possible to detect the corresponding Xenopus gene by low stringency hybridization of a Southern blot: the EcoRI digested Xenopus DNA showed two bands, one of 10 kb and the other of 7.4 kb; only the latter was analyzed in the present study. On the basis of these observations, 100 pg of Xenopus DNA digested with EcoRI was separated on a preparative scale, and a strip containing 7.4 kb DNA fragments was excised from the gel. The recovered DNA, enriched for the NGF sequence, was cloned in the EcoRI site of the phage lambda g t l l . It could be approximately evaluated that the 7.4 kb fragments preparation represented about 1-2% of the total DNA. This enrichment allowed us to find four positive clones in 50,000 plaques. In all four positive recombinants the 7.4 kb insert was split in two pieces, one of 5.3 kb and the other of 2.1 kb, when digested with EcoRI. A possible explanation of this fact is that this EcoRI site is methylated in the Xenopus cells. Since only the 5.3 kb band maintained the hybridization to the chicken probe, only this has been analyzed. Figure 1 shows the restriction map of this fragment and the strategy used to sequence a portion of a HindIII-AccI subfragment of 2.3 kb, which was still recognized by the chicken probe. The same figure also indicates the Hinfl-Hinfl fragment of 328 bp, covering part of the sequence for mature NGF, which has been used as a probe for Southern and Northern hybridization experiments.

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Sequence Analysis and Comparison With NGF of Other Species Sequencing of the HinfI -AccI fragment described in Figure 1has shown that it contains the last exon of the NGF gene. This encodes part of the N-terminal cleaved sequence of the prepro-NGF, all the mature protein and extends into the 3' untranslated region. In Figure 2 the sequence of the last exon of the gene and the flanking regions together with the derived aminoacid sequence are shown. Comparison of the nucleotide sequence of mature NGF of Xenopus to those of snake, chicken, mouse, and man indicates a conservation of 70%, 80.5%, 74.5%, and 78%, respectively; most of the differences are found in silent positions (not shown). Some stretches (underlined in Fig. 2) of the 3' untranslated region are highly conserved with respect to mouse, human, and chicken (Ebendal et al., 1986). The characteristic presumptive polyadenylation signal ATTAAA, found in other vertebrate NGF mRNAs (Scott et al., 19831, is also present in Xenopus. Figure 3 compares the aminoacid sequence of Xenopus prepro-NGF with those of mouse, human, bovine, rat, chicken, and snake (Scott et al., 1983; Ullrich et al., 1983; Meier et al., 1986; Whittemore et al., 1988; Ebendal et al., 1986; Selby et al., 1987a,b). It can be observed that a great part of the mature NGF molecule is very conserved in all species, but also that Xenopus shows a greater homology with higher vertebrates than with the snake. The cysteine, histidine, and tryptophan residues are strictly conserved. In the prepropeptide, that is the portion which is cleaved at maturation, small stretches of well-conserved sequence alternate

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Fig. 1. Restriction map of the genomic segment containing the last exon of the Xenopus NGF gene. The 2.3 kb HindIII-AccI fragment was subcloned and sequenced with the strategy indicated by the arrows. Only part of the intron which spans upstream of the last NGF exon has been sequenced. Mature NGF (open box), prepropeptide (shaded box),

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3' untranslated region (dashed box). The position of the fragment used as homologous probe is indicated. Restriction enzyme abbreviations are as follows: A = AccI; D = DdeI; E = EcoRI; H = HinfI; Hd = HindIII; R = R s d .

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F. CARRIER0 ET AL. 115 ....TGAGATTCACTAGAACAGATATCAGATCAGATATTCATATCTTTAGTAATTGCGTTCAGTAGCATTCTCTCTTAAATTAGATAAGCAGATGTTTGCGTTTGTATTTCTACCTATT -118 V a l Asp A r g V a l Met Ser Met Leu T y r T y r Thr Leu Leu I l e A l a l l e Leu l l e Ser V a l TACACATGCATAACATATTTATCTTTTTTTATTTTTTAG,GTG GAT AGA GTA ATG TCC ATG TTG TAC TAC ACT TTG TTG ATA GCA A T 1 CTC ATC AGC GTA

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Phe G l u Thr Lys Cys Arg Asp P r o Lys P r o V a l Ser Ser G l y Cys A r g G l y l l e Asp A l a L y s His T r p Asn Ser T y r Cys Thr Thr Thr T T T GAG ACC AAA TGC AGG GAC CCA AAG C C A GTT TCA AGT GGA TGC CGT GGG A T 1 GAT GCA AAG CAT TGG AAC TCT TAT TGT ACC ACC ACA

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H i s Thr Phe V a l L y s A l a Leu Thr Met G l u G l y L y s Gln A l a A l a T r p A r g Phe I l e A r g l l e Asp Thr A l a Cys V a l Cys V a l Leu Ser CAC ACC T T T G T C AAA G C A T T A ACA ATG G A A GGG AAG CAA GCA GCA TGG AGA TTC ATA C G G A T T GAT ACA GCA TGT GTC TGT GTG CTA AGC

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ATACTGCAAAAGAGAGACATTATTTATTATTAAACAATTTTTAAAATTCTGTGTTTGTTGTTTTTCAAGTCTAGTTTTATGT __

82

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Fig. 2. Nucleotide sequence ofXenopus NGF last exon and deduced aminoacid sequence. The exon-intron boundary is indicated by the arrow. Mature NGF begins a t aminoacid 1.Groups of basic aminoacids are boxed and a possible glycosylation site is overlined. Conserved

sequences in the 3’ untranslated region are underlined, and the characteristic presumptive polyadenylation signal is underlined with double line.

with largely divergent regions: conserved features among species are two basic blocks, one located next to the mature protein and the other around position -42, the presumptive signal peptide sequence (-105 to -118), and the three prolines at position -26 with ten upstream aminoacids. At variance with higher systems, both Xenopus and snake have a region rich in acidic aminoacids between -13 and -24. A particular feature of Xenopus as compared to other systems is a deletion(s) of some aminoacids in a poorly conserved area where the alignment is difficult and arbitrary.

sites, the hybridization on the 10 kb band is consistent with the observation that most genes detected so far in the Xenopus laevis genome are present in two copies and is in accordance with the hypothesis of a total genome duplication during the evolution of Xenopus laevis (Bisbee et al., 1977).

Analysis of the NGF Gene Copy Number in Xenopus The Southern blot analysis described above, where a heterologous chicken probe was used, resulted in two hybridization bands. These could be due to the presence of two gene copies or to the presence of an EcoRI site within the portion of the NGF gene spanned by the probe. To distinguish between these two possibilities, genomic Xenopus DNA was digested with EcoRI and analyzed by Southern blot hybridization with the homologous probe of 328 bp. The hybridization, carried out at high stringency, revealed the presence of two bands, one of 10 kb and the other of 7.4 kb, the same bands obtained with the chicken probe (see above). Since the Xenopus 328 bp fragment is a derivate of the cloned 7.4 kb fragment and does not contain EcoRI

Expression of the NGF Gene in Xenopus Oocytes and Embryos In order to find out about the onset of NGF gene expression during development we analyzed the accumulation of NGF mRNA in oocytes and embryos. Poly(A)+ RNA was prepared from defolliculated oocytes of stage I, 11, and VI and from embryos of different stages. These developmental stages seemed suitable to reveal a possible maternal supply and a stage-specific expression during the formation of the nervous system. Amounts of poly(A)+ RNA equivalent to 75 oocytes and embryos were loaded on a Northern gel, blotted, and hybridized with the Xenopus NGF probe. As shown in Figure 4, NGF mRNA is detectable in oocytes of stage I, it increases at stage 11,and remains constant up to stage VI. This mRNA is still present in stage 5 embryos, becomes undetectable around gastrulation, but appears again at neurulation and increases progressively in the following stages. For comparison the same filter was rehybridized with a probe for ribosomal

XIINOPUS NGF GENE

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Fig. 3. Alignment of pre-pro NGF aminoacid sequence of Xenopus, X, and other species: M, mouse (Scott et al., 1983);H, human (Ullrich et al., 1983);B, bovine (Meier et al., 1986);R, rat (Whittemore et al., 1988);C, chicken (Ebendal et al., 1986);S, snake (Selby et al., 1987a). The alignment is relative to mouse NGF whose aa. numeration is here maintained. Mature NGF begins at aminoacid 1.For each species only

differences are indicated. Dashed lines denote unknown sequence; dots represent gaps to obtain the best homology. Groups of basic residues are boxed. Note that due to the deletion(s) the Xenopus prepropeptide coded for by the third exon is made of 118 aminoacid,see also Figure 2.

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F. CARRIER0 ET AL. Embryos

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S t a g e s Fig. 4. Accumulation of NGF mRNA during oogenesis and embryogenesis. Poly(A)+ RNA equivalent of 75 oocytes and embryos was loaded on a Northern gel, blotted, and hybridized to the radioactive Xenopus NGF probe. The first lane a t left (ov) was overloaded with ovary poly(A)+ RNA. Positions of size markers (kb) are indicated on the right: 4.1 and 1.8 kb correspond to Xenopus 28s and 18s rRNA, respectively.

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protein S1 (not shown); the typical developmental pattern of ribosomal protein mRNA accumulation was observed (Pierandrei-Amaldi et al., 1982). The possibility that the NGF mRNA found in oocytes may come from residual follicular cells contaminating the preparation is ruled out by the fact that it is present in early embryos. In fact this RNA can be only of maternal origin as transcription is silent in this developmental period. In order to have an idea of the relative abundance of the NGF mRNA among oocyte mRNAs, a quantitation was attempted by comparison with the same filter rehybridized with a probe for S1r-protein mRNA whose abundance is known. By using similarly labeled probes, normalizing for the exposure time, and considering that the mRNA for one r-protein is about 0.1% of oocyte mRNAs, it can be calculated that the amount of NGF mRNA is between 0.01 and 0.001% of the oocyte mRNA population, indicating that it belongs to the low abundance class of mRNAs. The size of the NGF mRNA in oocytes and embryos is about 1.2 kb, as determined by comparison with RNA markers, a size similar to NGF mRNA of other species. A slowly migrating hybridization band, larger than 5 kb, can also be observed after gastrulation and is maintained during the following stages, but is not found in oocytes even when a large amount of RNA was loaded on the gel (see first lane in Fig. 4).The possibility that this band is due to a trapping of the probe by residual 28s rRNA is ruled out by the fact that it migrates more slowly than 28s rRNA, as observed after rehybridization of the same filter with a rRNA probe; moreover it does not appear after hybridization with other probes (for instance r-protein) even when the film is overexposed (not shown). The nature of this band has yet to be elucidated.

Polysome/mRNP Distribution of NGF mRNA

Once established that NGF mRNA is present in early and late embryogenesis, and surprisingly also in VI oocytes, it was interesting to know how the NGF is synthesized and accumulated. To have this information we analyzed the engagement of NGF mRNA with polysomes, as an indication of the synthesis of the 4 protein. Defolliculated oocytes of stages 11, 111, and VI and embryos of stages 4,7, and 15 were homogenized and the cytoplasmic extracts were separated on polysome gradients as described in “Materials and Meth7 ods.” The poly(A)+ RNA purified from polysomes and mRNPs was loaded on Northern gel and hybridized to 15 the NGF probe. Figure 5 shows that in stage I1 oocytes the majority of NGF mRNA is located on polysomes indicating an active synthesis of the protein, whereas NGF rp-Sl later in oogenesis it becomes progressively excluded Fig. 5. PolysomeimRNP distribution of NGF mRNA in oocytes and from polysomes. The same Figure shows that after embryos. Cytoplasmic extracts from oocytes and embryos of different fertilization the maternal NGF mRNA passed to the stages were fractionated on sucrose gradients. Poly(A)+ RNA was prepared from the polysome and mRNP regions and analyzed by embryo is still excluded from polysomes and then Northern blot hybridization with the NGF probe. The RNA loaded on undergoes degradation (see also Fig. 4, stage lo), each lane corresponds to the same number of individuals. As a control whereas about 50% of the newly synthesized RNA the same filter was hybridized to a probe for r-protein S1. present at stage 15 is loaded on polysomes indicating 0 0

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Fig. 6. Analysis of NGF in oocytes and embryos by antibodies. a: The cytoplasmic soluble extract equivalent of 10 oocytes (Ooc.) and embryos (Emb.) were loaded on protein gels, western blotted and incubated with affinity purified antibodies versus mouse-NGF. Positions of the molecular weight markers are indicated. b 35S-methionine-labeled translation product directed by synthetic NGF mRNA (t);the band a t the bottom is globin synthesized by the rabbit reticolocyte lysate alone (-). c: Three identical oocyte samples were loaded on a gel and transferred onto a filter. This was cut in three strips that were separately incubated with antibodies. The first was incubated with antibodies alone, the others also with 2 pg and 8 pg of mouse NGF, respectively, as competitor (Comp.NGF). The last two strips were developed for a long time and this made more evident some background which partially appeared also in the preimmune control (not shown). Arrows indicate the size markers from top to bottom. The stars a t right of each panel mark the 32 kD band.

that the protein begins to be synthesized. For comparison we have hybridized the same filter with the r-protein S1 probe which showed the expected distribution pattern of r-protein mRNA. In particular, at variance with NGF mRNA, the newly synthesized S1 mRNA of stage 15 embryos is mainly found in mRNPs, due to the translational control characteristic of this class of mRNAs which allows efficient synthesis only at later stages (Pierandrei-Amaldi et al., 1982). The real engagement of NGF mRNA with polysomes was tested by EDTA treatment of the extract followed by centrifugation on EDTA gradients. It was observed that the dissociation of polysomes by EDTA was accompanied by a shift of the NGF mRNA toward the light part of the gradient (not shown) indicating that this mRNA is engaged in protein synthesis.

Accumulation of the NGF Protein Affinity purified polyclonal antibodies raised against mouse NGF were used in order to analyze the accumulation of NGF in oocytes and early embryos. Soluble proteins from the same equivalent of defolliculated oocytes and embryos were loaded on protein gels and analyzed on western blots. The immunoreaction re-

319

vealed a band of the apparent size of 32 kD that is present in stage I1 and VI oocytes, is maintained up to stage 8 embryos, and disappears within stage 14; later on, at stage 30, the band seems to appear again (Fig. 6a). The band of 32 kD does not coincide with any stained band in the gel nor does it appear when using preimmune serum (not shown). Sometimes a faint band of about 17 kD, very difficult to photograph, is detectable. Two monoclonal antibodies anti-mouse NGF also recognize the 32 kD protein (not shown). In order to verify the specificity of the reaction, competition experiments were performed by adding mouse NGF as a competitor to the immunoreaction (Fig. 6c)..It can be observed that a 32 kD band is efficiently competed by mouse NGF. In this experiment antibodies and NGF were not preincubated in advance, but were added directly to the filter. Thus some antibodies could reach the antigen on the filter, resulting in a residual reaction in the competed samples. These results suggest that the protein revealed by the antibodies could be the putative precursor form of Xenopus NGF. Furthermore the last exon of the NGF gene was cloned,into a SP6 transcription vector that allows the in vitro synthesis of an NGF mRNA, which encodes for the NGF precursor including the presumptive signal peptide. This transcript corresponds with the shorter of the two principle transcripts found in mouse and human which utilizes as initiator the methionine immediately upstream of the presumptive signal peptide (Edwards et al., 1986). In vitro translation of this synthetic Xenopus NGF RNA generates a product which has the same electrophoretic mobility as the protein recognized by the antibodies (Fig. 6b). It must be pointed out that the expected size of the in vitro product, calculated from the sequence, is 26,414 daltons, but that it migrate as 32 kD by comparison with standard markers. A similar discrepancy was reported for the mammal NGF precursor and it was concluded that it migrates aberrantly in SDS-polyacrylamide gels as occurs also for other proteins (Edwards et al., 1988).

DISCUSSION To undertake the study of the expression of the NGF gene in Xenopus development it was important to have clones for the corresponding homologous sequences. For this purpose we have isolated and characterized a Xenopus genomic DNA fragment which contains the entire last exon of the NGF gene; this last exon, as is the case for other vertebrates, codes for the complete mature NGF and most of the prepropeptide including the putative signal peptide; it contains also the 3’ untranslated region and extends beyond the polyadenylation signal.Southern blot analvsis of genomic DNA indicated that the NGF gene Ts preseit in two copies in the genome of Xenopus laeuis, as occurs also for most other genes as a consequence of a whole genome duplication which took place in this species about 30 million years ago (Bisbee et al., 1977).

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Comparison of the aminoacid sequences of mature NGF of Xenopus and of other species shows a high conservation. The cysteine, histidine, and tryptophan residues are strictly conserved, suggesting an essential role for the biological activity of the molecule (Frazier et al., 1973). In contrast in the prepropeptide, large divergent regions alternate with some short regions which are very conserved. These are the presumptive signal sequence and the basic sites probably required for the correct processing of the precursor molecule. However, in spite of the belief that conservation is essential for function, in a recent report it was shown that replacement by site-directed mutagenesis of some very conserved positions in the NGF molecules did not significantly modify the biological activity as compared to the normal molecule (Ibanez et al., 1990). It seemed interesting to study whether NGF has some role in neural differentiation during early embryogenesis and in what way. An advantage of the Xenopus system, as compared with higher vertebrates, where studies on NGF have been mostly carried out, is the possibility of investigating with ease early embryonic events and of interfering with normal development by embryo manipulation. As a first approach to study this problem we have analyzed the accumulation of NGF mRNA in developing oocytes and embryos. Quite unexpectedly we have observed that NGF mRNA is present in defolliculated oocytes: it is already detectable at stage I, it increases in amount at stage 11, and it is maintained nearly constant at stage VI. At fertilization this NGF mRNA is passed to the embryo and it disappears before gastrulation. New NGF mRNA appears again around neurulation as a product of embryonic transcription which is activated at midblastula (Davidson, 1986; Newport and Kirschner, 1982). The NGF mRNA keeps increasing after neurulation in developing embryos, where the sensitivity of Xenopus tadpole nerve cells to the NGF molecule has been established (Levi-Montalcini and Aloe, 1985). NGF mRNA belongs to the low abundance class of the oocyte mRNA and its length is about 1.2 kb. A higher band which hybridizes with the NGF probe was reproducibly observed in embryos, but not in oocytes. The nature of this RNA is not yet known: it might represent a RNA precursor of NGF, as also observed in other systems (Heumann et al., 1984; Ebendal and Persson, 1988). To find out if the NGF mRNA was utilized for protein synthesis, particularly in the oocytes where the presence of a growth factor supposedly specific for the nervous system appears rather peculiar, we have examined its association with polysomes in oocytes and in early embryos, considering this as a bona fide indication of NGF synthesis. The experiments have shown that the NGF mRNA is fully engaged with polysomes in early oogenesis and progressively dissociates from them during oocyte growth. At fertilization the mRNA is passed on to the embryo, remains excluded from polysomes and is degraded before gastrulation. In contrast the new NGF mRNA of embryonic synthesis,

present around neurula stage, is associated with polysomes in agreement with the active neural development which takes place at this stage. These observations suggest that some NGF protein is produced in oocytes. In fact using antibodies against mouse NGF it was possible to identify on western blots a strongly positive band of the apparent size of 32 kD; this band has the same mobility as the in vitro translation product of the transcript relative to the third exon of the NGF gene. These facts are in favor of the presence of a precursor form of NGF which is accumulated in the oocytes and is passed to the embryo at fertilization, suggesting a maternal role for this protein. Sometimes a smaller form, barely detectable, was observed: it is possible that the NGF, accumulated as a precursor in the oocyte, is matured into the active form at very low levels or it is rapidly turned over. A similar behavior is observed for Vgl, a protein related to the TGF-P family of small secreted growth factors, which is found in Xenopus oocytes and embryos as a full-length molecule, whereas the small active molecule can be hardly detected (Tannahill and Melton, 1989). It can thus be speculated that NGF could have some activity in early neural differentiation: if this is the case the protein, maternally inherited in an immature form, could be matured in the embryo, possibly starting its function when responding cells begin to express specific receptors. Later on, the NGF activity on nerve cells would be assured by the embryonic expression of the NGF gene here described. Another possibility, which cannot be excluded, is that this protein exerts some growth factor-like function in the oocyte and narrows its range of activity when responding cells become competent. Since a large number of agents such as insulin (Maller and Koontz, 1981) and insulin-like growth factors (Bauliau and Schorderet-Slatkine, 1983) can stimulate the progesterone effect on oocyte maturation, the fact that NGF can intervene in this oocyte function seems possible. On the other hand it has been recently reported (Sehgal et al., 1988) that treatment of Xenopus oocytes with high concentrations of human NGF did not stimulate progesterone-induced maturation; NGF had no effect also when it was added to oocytes which were expressing on their surface the human NGF receptor synthesized by human mRNA injected several hours before. However the presence of human NGF receptors itself potentiates the ability of progesterone to induce maturation. Our observation that oocytes accumulate and translate NGF mRNA, thus producing an endogenous supply of this growth factor, could provide an explanation to the results above described, namely that oocyte NGF could interact with the synthesized receptors. In line with our results the presence of NGF mRNA and protein in male germ cells of mouse and rat was reported (Ayer et al., 1988). Although at the moment we do not have a physiological explanation for the presence of NGF in oocytes and in early embryos, we consider it unlikely that it is the produce of aspecific expression. In fact it is known that

XENOPUS NGF GENE the complexity of Xenopus oocyte RNA is high but not so high, compared to the complexity of somatic RNA, to justify a random expression of genes in oocytes. Some aspecific transcription was reported at lambrush stages, but transcripts were not stabilized and disappeared in the following stages (for a detailed discussion, see Davidson, 1986). Moreover it was shown that some somatic mRNA sequences, such as globin and vitellogenin mRNAs, are absent or at least 10-100 fold less abundant in the oocyte than an average rare oocyte sequence (Schaefer et al., 1982). Further work is necessary to find out if the NGF has a physiological role in the oocyte and in the early embryo. This can be considered possible according to the concept that hormones, growth factors, and other hormone-like agents and their receptors, whose activity is well established atflaterstages of life, may also be mediators of crucial events in early embryogenesis (De Pablo and Roth, 1990 and reference therein). It is now important to analyze the spatial distribution of NGF throughout oogenesis and embryogenesis and to study the effect of overexpression and underexpression of the NGF gene.

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