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Retroviral promoter-trap insertion into a novel mammalian septin gene expressed during mouse neuronal development
Mechanisms of Development 86 (1999) 183±191
Gene expression pattern
Retroviral promoter-trap insertion into a novel mammalian septin gene expressed during mouse neuronal development Jing-Wei Xiong, Amy Leahy, Heidi Stuhlmann* Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029, USA Received 22 February 1999; received in revised form 4 May 1999; accepted 10 May 1999
Abstract We have characterized a retroviral promoter-trap insertion into a novel mammalian septin gene, Sep3. Its predicted amino acid sequence shares signi®cant homology to that of Saccharomyces cerevisiae CDC3, CDC10, CDC11, CDC12, the Drosophila genes Pnut, Sep1, Sep2, and the mammalian genes BH5, CDC10, Nedd5, Diff6, and Sep2, which are implicated in cytokinesis and cell polarity. Sep3 encodes a protein of 465 amino acids, and contains an evolutionary conserved ATP/GTP-binding motif, two coiled-coil domains, and a highly hydrophobic domain at the C terminus. Alkaline phosphatase reporter gene expression in transgenic embryos was ®rst detected at E8.5 in the neural fold, and high levels of expression continued throughout embryogenesis in the neural tube and brain. In addition, a low level of transient expression was detected in the somites, gut, and branchial arches of mouse embryos. Overall, reporter gene expression recapitulated Sep3 mRNA expression during mouse embryogenesis. In adults, Sep3 transcripts were only detected in the brain and testis. Zoo blot analysis revealed that Sep3-related sequences exist in several vertebrate species including zebra®sh, frog, chicken, mouse and human. Consistent with the retroviral insertion into the 3 0 UTR of the Sep3 gene, no obvious phenotypes associated with the promoter trap were detected in transgenic embryos or adult mice. In summary, we report the ®rst isolation of a novel full-length Sep3 cDNA and extensive characterization of its expression during mouse embryogenesis and in adult tissues. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Septins; Sep3; Promoter trap; Alkaline phosphatase reporter gene; Genetic screen; Neuronal development; Mouse embryogenesis; ES cells; Embryoid bodies; Transgenic mice
1. Results 1.1. AP expression in transgenic 2-86 embryos is largely restricted to the developing CNS In an approach to identify genes involved in early mouse development, we have performed an entrapment vector screen in ES cells and mouse embryos (Xiong et al., 1998). ES cell clone 2-86 was identi®ed as a candidate clone based on the spatially and temporally restricted expression of the alkaline phosphatase (AP) reporter gene during ES cell differentiation into embryoid bodies (Xiong et al., 1998). To examine AP reporter expression in vivo and to determine if the promoter-trap insertion caused a mutant phenotype, we established a transgenic mouse line by using the diploid embryo-ES cell aggregation technology (Nagy et al., 1993; Xiong et al., 1998). Mice hetero- or homozygous for the 2-86 insertion were viable * Corresponding author. Tel.. 11-212-241-9413; fax: 11-212-8609279. E-mail address: [email protected] (H. Stuhlmann)
and fertile, and did not show overt abnormalities. Heterozygous males carrying the 2-86 insertion were bred with wild-type CD-1 females. Staged embryos were dissected out of their deciduas and subjected to AP staining. The earliest time point of AP expression in whole-mount embryos was detected at E8.5 in a region where folding of the neural plate begins. In E8.5 to E9.0 embryos, strongest AP expression was detected in the anterior neural tube and posterior hindbrain and weaker expression in other regions of the developing CNS, the somites and branchial arches (Xiong et al., 1998). Expression appeared restricted to dorsal aspects of the neural tube that may include neural crest cells, and was not detected in cephalic mesenchyme, foregut, or branchial arches (Fig. 1A,B). In transverse sections through the posterior part of embryo, AP expression was detected in the neural tube, notochord, the dorsal part of the hind gut, and somites (Fig. 1C,D). In wholemount E10.5 embryos, strong AP expression was found throughout the brain, in the optic vesicle, and the spinal cord, and weak expression in the somites and branchial arches (Xiong et al., 1998). Strong AP expression was
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Fig. 1. AP reporter gene expression in 2-86 transgenic embryos. Transgenic embryos were isolated, ®xed and stained for AP, post-®xed, embedded in paraf®n, and sectioned. (A±D) Transverse sections of E8.5 embryo through head region (A), foregut and branchial arches (B), hind gut (C), and posterior trunk (D), respectively. (E,F) Transverse sections of E9.5 embryo through optic vesicle (E), and Rathke's pouch (F). Abbreviations: BRA, branchial arch; CM, cephalic mesenchyme; E, epidermal ectoderm; FG, fore gut; HG, hind gut; NE, neural epithelium; NT, neural tube; OV, optic vesicle; RP, Rathke's pouch; SOM, somites.
detected in transverse sections through the head region, predominantly in the dorsal epithelium of the optic vesicle, and apparently localized to the future lens epithelium, the overlying corneal ectoderm and surrounding mesenchyme (Fig. 1E). Expression was also detected in Rathke's pouch region (Fig. 1F), the brain and spinal cord (not shown). 1.2. Molecular cloning of genomic sequences ¯anking the 286 insertion and Sep3 cDNA To understand the 2-86 insertion on the molecular level, we cloned a 9.4 kb 5 0 EcoRI-EcoRI fragment, and a 2.7 kb
3 0 EcoRI-EcoRI fragment by using the supF complementation approach (Xiong et al., 1998; see also Fig. 2A). A 3.0 kb 5 0 NheI-NheI fragment, and a 0.8 kb 3 0 KpnI-EcoRI fragment, containing mostly ¯anking genomic DNA, were subcloned and used as probes for southern analysis to verify that the genomaic clones corresponded to the 2-86 insertion (Fig. 2A). Both probes detected a 6.7 kb pre-integration fragment in EcoRI-digested, genomic DNA from R1 ES cells, clone 2-86, and another unrelated clone, 2-51, with a promoter-trap insertion. In addition, the 3 0 KpnI-EcoRI probe hybridized to a 3.4 kb fragment in DNA from clone 2-86, and DNA from R1 cells mixed with 10 ng of cloned
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genomic 3 0 EcoRI-EcoRI DNA, but not in DNA from clone 2-51 or R1 cells (Fig. 2A). Reprobing the ®lter with a neospeci®c probe detected the same 3.4 kb genomic fragment (not shown). The 5 0 NheI-NheI probe detected a 9.4 kb fragment in DNA from clone 2-86, but not in DNA from clone 2-51 or R1 cells (Fig. 2A). Since the design of the PT-I RES-AP vector predicted insertion into an exon of a targeted endogenous gene, both ¯anking regions should contain exonic sequences (Gossler and Zachgo, 1993; Xiong et al., 1998). The 0.8 kb 3 0 KpnIEcoRI fragment was used as a probe to screen a day-11.5 mouse total embryo cDNA library (Clonetech), and ®ve independent cDNA clones were isolated, ranging from 0.5 to 2.0 kb in length. Sequencing revealed that they shared signi®cant homology with several members of the septin family. Clone #12-1 was the longest cDNA clone, and its sequence corresponds to nucleotides 470±2455 of the fulllength Sep3 gene. To obtain a full-length cDNA clone, a subclone of this cDNA, #12-1 B (corresponding to nucleotides 470±820), was used as a probe to screen an adult brain cDNA library. Several cDNA clones containing the complete coding regions of Sep3 were isolated. The longest cDNA clones contained 2455 bp of sequence (Fig. 2B). Alignment of the 3 0 ¯anking genomic DNA sequence with that of Sep3 cDNA revealed that the retroviral insertion in clone 2-86 mapped to the 3 0 untranslated region of this gene (underlined, bold nucleotides TG at 1821±1822 in Fig. 2B). Consistent with the 2-86 insertion in the non-coding 3 0 region of the Sep3 gene, no obvious phenotypes were detected in 2-86 transgenic embryos or adult mice (see above).
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al., 1992; Kinoshita et al., 1997), with 99% identity to human NED5 in its amino acid sequence. Genetic and molecular analysis of septins from yeast, Drosophila and mammals revealed that septins are required for cytokinesis (Longtine et al., 1996) as well as for establishment of cell polarity (Hsu et al., 1998). Alignment of the predicted SEP3 amino acid sequences and that of several other septin proteins uncovered that overall sequence identity ranged from 36% for yeast CDC3 to 50% for mouse NEDD5 (Fig. 2C, and not shown). The putative SEP3 protein contains a highly conserved ATP/GTP-binding domain at the N terminus, containing the P-loop sequence GQSGLGKS (Fig. 2B,C) (Saraste et al., 1990; Neufeld and Rubin, 1994) which may be involved in protein assembly or regulation (Longtine et al., 1996). Based on the Kyte±Doolittle analysis in the MacVector program, a highly hydrophobic region of 60 amino acids is located in the carboxyl terminal region (amino acids 360±420). Two putative coiled-coil domains were identi®ed in two regions of SEP3 (amino acids 129±154 and 175±237, respectively; underlined in Fig. 2B), using the World Wide Web server of the Swiss Institute for Experimental Cancer Research for prediction and analysis of coiled-coil structures (Lupas, 1996). Overall homology between SEP3 and other proteins of the septin family are high except for their N- and C-terminal regions; however, homology among other mammalian septins is higher than between any of those and SEP3 (Fig. 2C, and not shown).
1.3. Sep3 is similar to cell cycle control genes
1.4. Sep3 mRNA expression is found in the developing CNS and somites and is essentially identical to AP expression in transgenic embryos
The nucleotide sequence of the 2455 bp Sep3 cDNA revealed a single open reading frame of 1395 bp that encodes a putative polypeptide of 465 amino acids with a predicted molecular mass of 51 kDa (Fig. 2B). Homology searches of current databases revealed that the putative SEP3 protein is a novel mammalian member of the septin family and shares signi®cant homologies to the yeast cell cycle control proteins CDC3, CDC10, CDC11, and CDC12 (Hartwell, 1971), the Drosophila proteins Pnut, Sep1, and Sep2 (Neufeld and Rubin, 1994; Fares et al., 1995), as well as eight human and four mouse septin genes. The human genes are BH5 (GenBank accession #3290200); CDC10 (Nakatsuru et al., 1994); H5 (McKie et al., 1997; Zieger et al., 1997); KIAA0128 (Nagase et al., 1996); NED5 or KIAA0158 (Nagase et al., 1996); a putative septin (GenBank accession #e1167897); a gene related to Drosophila Sep2 (Nagase et al., 1996); and a CDC-related protein (GenBank accession #3283224). The mouse genes are BH5 (Kato, 1990), with 93% identity to human BH5 in its amino acid sequence; CDC10 (GenBank accession #e1250468); Diff6 (Nottenburg et al., 1990); and NEDD5 (Kumar et
RNA in situ hybridization analysis was used to determine the temporal and spatial distribution of Sep3 mRNA during mouse embryogenesis. A sense riboprobe did not show any speci®c signal (Fig. 3b, panel C). The antisense Sep3 probe detected expression primarily in the developing CNS in E9.5±E13.5 embryos. At E10.5, strong signal was detected in the neural epithelium throughout the fore-, mid-, hindbrain, and the neural tube, and transiently in the surface ectoderm (Fig. 3a, panels A,B). Weaker expression was observed in the dorsal root ganglia, the somites, and gut (Fig. 3a, panels A,C, and not shown). No speci®c signal was detected in heart and liver. At E11.5 and E12.5, Sep3 expression was observed primarily in neuronal tissue and dorsal root ganglia (not shown). At E13.5, high signal stayed on in all parts of the brain and the neural tube, and weaker signal was detected in the dorsal root ganglia (Fig. 3b, panels A,B). Highest Sep3 expression was detected in the outer layer of cells of the fore- and midbrain (Fig. 3b, panels A,B). In summary, Sep3 mRNA expression was located primarily in developing CNS and PNS and was essentially identical to AP expression observed in 2-86 transgenic embryos (compare Fig. 1 to Fig. 3).
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1.5. Sep3 transcripts are detected in mouse embryos and in adult brain and testis To determine the size and distribution of Sep3 transcripts, Northern blot analysis was performed on total RNA from staged embryos between E7.5 and E16.5, and from adult organs (Fig. 4). Two major Sep3-speci®c transcripts of about 6.0 kb and 2.4 kb in length were detected at increasing levels in embryos between E8.5 and E13.5, but not in E7.5 embryos (Fig. 4) or ES cell RNA (not shown). The same two transcripts were detected in brain, but not in any other adult mouse tissues examined. The relative abundance of the two transcripts was similar in all sources of RNA. The smaller transcript of about 2.4 kb in length corresponded in size to the 2.45 kb Sep3 cDNA that we isolated. In adult testes, less abundant transcripts of 4.2 kb and approximately 0.8 kb length were detected. 1.6. Sep3-related sequences are conserved in vertebrates `Zoo blot' analysis was performed to determine whether Sep3-related sequences were present in other vertebrate or invertebrate species. Genomic DNA from zebra®sh, Xenopus, Gallus, mouse and human was digested with BamHI, EcoRI, or HindIII, respectively, and subjected to Southern blot analysis. Filters were hybridized at reduced stringency with a 2.45 kb Sep3 cDNA probe and washed at high stringency. One or two strong hybridizing fragments and several fragments with lower intensity were detected in the genomic DNA from mouse, human and chicken (Fig. 5). In DNA from lower vertebrates (Fig. 5) and sea urchin (not shown), between one and four bands with lower intensity were detected. These results suggest that the mouse Sep3 gene belongs to a small family of genes which share related sequences in other vertebrate species. 2. Discussion We report here on the identi®cation and characterization of the endogenous gene targeted by a promoter trap insertion, Sep3. Sep3 belongs to the evolutionary conserved family of septin genes, and its expression during mouse embryogenesis is located primarily in the developing CNS and PNS. Sep3 encodes a putative protein of 465 amino acids that shares about 36% to 50% overall amino acid sequence identity with known septins in S. cerevisiae,
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Drosophila, mouse and human. It shows several characteristic hallmarks of septins including an evolutionary conserved ATPase/GTPase motif and two coiled-coil domains. In addition, Sep3 contains a highly hydrophobic domain at its C terminus that is distinct from other septins. Current understanding of the biological functions of septins largely comes from analysis of the yeast CDC3/10/11/12 and the Drosophila Pnut mutants, all of which have distinct phenotypes in cytokinesis. Although several septin genes with signi®cant overall amino acid sequence homology are found in each species, each individual septin appears to play an important role rather than displaying functional redundancy or compensation in cytokinesis (Neufeld and Rubin, 1994; Longtine et al., 1996). So far, at least eight mammalian septin genes have been identi®ed and partially characterized. Original ts-lethal mutations including mutations in S. cerevisiae septin genes CDC3, CDC10, CDC11, CDC12, and the Drosophila Pnut mutant have shown defects in cytokinesis (Hartwell, 1971; Neufeld and Rubin, 1994). Furthermore, anti-septin antibodies localized septin gene products to the mother-bud neck in the yeast (Longtine et al., 1996), to the cleavage furrows and residual intercellular bridges in the ¯y (Neufeld and Rubin, 1994; Fares et al., 1995), and near the contractile ring and midbodies in the mouse, respectively (Kinoshita et al., 1997). EM studies suggested that the septin-containing structures are closely associated with the plasma membrane (Byers and Goetsch, 1976; Neufeld and Rubin, 1994; Fares et al., 1995; Kinoshita et al., 1997), and they fractionate largely with the crude membrane fraction (Longtine et al., 1996). RNA in situ hybridization analysis revealed that Sep3 expression is predominantly localized to the neural epithelium and to differentiating, post-mitotic neurons. Interestingly, it has been reported that other septins also display strong expression in the nervous system, e.g. Drosophila septins (Neufeld and Rubin, 1994; Fares et al., 1995), mouse Nedd5 and H5 (Kumar et al., 1992; Kinoshita et al., 1997; Kato, 1990, 1992), and four rat septins (KIAA0128, CDC10, NEDD5, and H5) that are associated with a sec6/8 complex (Hsu et al., 1998). What is the function of septins in these non-dividing neurons? It has been proposed that septins are involved in the generation of the leading edge of membrane extension as well as in establishing cell polarity both in yeast and animal cells (Longtine et al., 1996; Hsu et al., 1998). In particular, both the sec6/8
Fig. 2. Molecular cloning of the 2-86 retroviral insertion site and Sep3 cDNA. Proviral and ¯anking cellular sequences were isolated by using the supF complementation method (Xiong et al., 1998). Sep3 cDNA was isolated and sequenced. Sep3 cDNA and its predicted sequence were analyzed by using the MacVector program. (A) Left: schematic map of the 2-86 proviral integration site with the PT-IRES-AP vector ¯anking genomic DNA. The positions of EcoRI (E) restriction enzyme sites used for supF are indicated. A KpnI (K) and two NheI (Nh) sites were used for cloning of a ¯anking 5 0 I-NheI and a 3 0 KpnI-EcoRI genomic fragment. Right: genomic DNA was digested with EcoRI and transferred to nitrocellular membranes. The blot containing 10 mg R1 ES cell DNA mixed 10 pg cloned 3 0 EcoRI-EcoRI fragment, 10 mg DNA each from ES cell clone 2-86, ES cell clone and R1 ES cells were hybridized with a 0.8 kb [a- 32P] dCTP-labeled 3 0 KpnI-EcoRI fragment 3.0 kb [a- 32P]dCTP-#labeled 5 0 NheI-NheI fragment. The position of the 5 0 and 3 0 EcoRI fragments, and the preintegration EcoRI-EcoRI fragments are indicated by arrow heads. (B) Nucleotide and deduced amino acid sequence of the Sep3 cDNA. The ATPase/GTPase domain is indicated by asterisks, and the two coiled-coil domains are underlined. The 2-86 insertion site, located between nucleotides 1821 and 1822, is shown in bold.
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Fig. 2C. Alignment of SEP3 sequence from amino acid 5 to 443 (top) with the respective sequences from mouse H5, CDC10, NEDD5, DIFF6, and human SEP2.
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Fig. 3. RNA in situ hybridization analysis of Sep3 mRNA in E10.5 and E13.5 embryos. Wild-type embryos were isolated, embedded in paraf®n and sectioned. Sections were processed for RNA in situ hybridization, using anti-sense or sense Sep3 (nucleotides 814±2455) riboprobes. (a) Analysis of E10.5 embryos. (A) Dark-®eld view of a parasagittal section of a E10.5 embryo. High-power magni®cation of hind-brain (B) and dorsal root ganglia (C). (b) Analysis of E13.5 embryos. (A) Dark-®eld view of a parasagital section of E13.5 embryo hybridized with anti-sense Sep3 probe. Dark-®eld view of high-power magni®cation of fore-brain hybridized with anti-sense Sep3 probe (B), or sense Sep3 probe (C). Abbreviations: bra, branchial arch; cm, cephalic mesenchyme; drg, dorsal root ganglia; epe, epidermal ectoderm; fb, forebrain; hb, hindbrain; hrt, heart; mb, midbrain; nep, neural epithelium; nt, neural tube; ov, optic vesicle; rp, rathke's pouch; som, somites.
Fig. 4. Expression of Sep3 mRNA in embryos and adult organs. 20 mg each of total RNA was resolved, transferred, and hybridized with a [a- 32P]dCTPlabeled 2.45 kb Sep3 cDNA fragment. After removal of the probe, the ®lters were re-hybridized with a [a- 32P]dCTP-labeled mouse GAPDH plasmid. (A) RNAs from staged CD-1 embryos between E7.5 and E16.5. (B) RNAs from adult organs of CD-1 mice. LIV, liver; SPL, spleen; KID, kidney; GUT, gut; MUS, muscle; LUN, lung; HRT, heart; THY, thymus; BR, brain; OVA, ovary; TES, testis.
Fig. 5. Southern `zoo blot' analysis of genomic DNA from different vertebrate species. Genomic DNA from different species was digested with BamHl (B), EcoRI (R), or HindIII (H), resolved on 0.8% agarose gels, transferred to nitrocellulose membranes, and hybridized to a [a- 32P]dCTP-labeled Sep3 probe. The ®gure is a composite of X-ray ®lms exposed for 1 day (DNA from chicken, mouse, human), and 4 days (DNA from zebra®sh, Xenopus), respectively.
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complex and septin ®laments are implicated in traf®cking vesicles and organizing proteins at the plasma membrane of neurons (Hsu et al., 1998). It will be of interest to determine whether SEP3 is involved in formation of the septin ®lament and sec6/8 complexes in neurons as well. The promoter-trap approach has been successfully applied to `tag' and to inactivate cellular genes in mouse ES cells and embryos (Gossler et al., 1989; Friedrich and Soriano, 1991). In this report, we have identi®ed Sep3 as the endogenous gene targeted by the 2-86 insertion. Consistent with its insertion into the 3 0 UTR of the Sep3 gene, no obvious phenotype was detected in transgenic embryos and adult mice derived from the 2-86 ES cell clone. In the future, loss-of-function analysis of Sep3, together with immunohistochemical localization of SEP3 in the developing CNS, will allow us to dissect its function during early mouse development. 3. Experimental procedures 3.1. Generation of ES cell aggregation chimeras and transgenic mice Clone 2-86 ES cells were used to generate germ-line chimeras by employing the ES cell-diploid embryo aggregation method (Nagy et al., 1993; Xiong et al., 1998). Three highly chimeric males transmitted the provirus through the germline. Inbred mice heterozygous and homozygous for the insertion were generated by crossing F1 founder males with 129/Sv females or b y intercrossing of heterozygous F1 males and females and genotyped by using a 0.8 kb KpnIEcoRI 3 0 ¯anking fragment as a probe (Fig. 2A). 3.2. AP staining of whole-mount embryos and histological sections Staged embryos were dissected from their deciduas of pregnant CD-1 females that were mated with heterozygous 2-86 males. Mouse embryos and extraembryonic membranes from stages E7.5±E13.5 were ®xed, heat-treated for 30 min at 708C, and subjected to AP staining using BM purple BCIP/NBT substrates (Boehringer Mannheim) as described (Xiong et al., 1998). Whole-mount stained embryos were post-®xed, embedded in paraf®n, and sectioned by conventional methods (Hogan et al., 1994). Photographs were taken under a Nikon SMZ-U stereomicroscope, or a Zeiss Axioskop microscope. 3.3. Molecular cloning of genomic DNA ¯anking the retroviral insertion and cDNA Genomic DNA containing retroviral vector, 5 0 and 3 0 ¯anking genomic sequences from clone 2-86 were isolated by supF complementation as described previously (Xiong et al., 1998). A 0.8 kb KpnI-EcoRI fragment containing 109 bp of 3 0 Mo-MLV long terminal repeat (LTR) and about 700 bp
3 0 ¯anking genomic sequences was used as a probe to screen an E11.5 mouse embryonic cDNA library (Clontech). The two longest overlapping clones, #8-3-1, and #12-1, were subcloned and sequenced on both strands by using an automatic DNA sequencer (ABI50, Utah State University). Resulting cDNA sequences lacked most of 5 0 sequences, and corresponded to nucleotides 470±2455 of Sep3 gene. In order to obtain a full-length cDNA clone, the most 5 0 EcoRI-EcoRI fragment of clone #12-1, #12-1B, was used as a probe to screen an adult mouse brain cDNA library (Stratagene). The three longest clones, #3, #11, and #12, were subcloned and sequenced as described above. All three clones contained the full-length coding region of the Sep3 gene. 3.4. RNA in situ hybridization Sep3 cDNA clone #12-1 A (corresponding to nucleotides 814±2455) was used as a template for the synthesis of sense and antisense riboprobes by RNA in vitro transcription. In situ hybridization to paraf®n-embedded embryo sections was performed as described previously (Leahy et al., 1999) 3.5. RNA isolation and Northern hybridization analysis Total RNA was prepared from staged embryos and adult mouse tissues by using a modi®cation of the guanidiniumCsCl method as described (Xiong et al., 1999). 20 mg samples of RNA were electrophoresed on 1% agaroseformaldehyde gels and transferred in 20 £ SSC to nitrocellulose membranes (Optitran, Schleicher and Schuell). Filters were hybridized at 428C in 50% formamide with 1 £ 107 cpm of a random hexamer-primed, [a- 32P]dCTPlabeled 2.45 kb fragment containing the full-length Sep3 cDNA. Filters were washed at 508C in 0:2 £ SSPE (36 mM NaCl; 2 mM NaPO4, pH 7.7; 0.2 mM EDTA), 0.1% SDS and exposed for 1±2 days. To control for RNA loading, ®lters were stripped and hybridized with a [a- 32P]dCTPlabeled mouse GAPDH cDNA plasmid. 3.6. Southern analysis of genomic DNA and `zoo blot' analysis Preparation of high molecular weight DNA, digestion with restriction endonucleases, and southern analysis was performed as described previously (Xiong et al., 1999). For analysis of the 2-86 integration site, ®lters were hybridized with a 0.8 kb 3 0 KpnI-EcoRI fragment, or a 3.0 kb 5 0 NheINheI fragment, respectively. For `zoo blot' analysis, ®lters were hybridized to 2 £ 107 cpm of a [a- 32P]dCTP-labeled 2.45 kb full-length Sep3 cDNA under reduced stringency conditions (358C in 50% formamide, 5 £ SSC, 2 £ Denhardt's, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA). Blots were washed at increasing stringency, with the ®nal washes at 658C in 0.2% £ SSC, 0.1% SDS for 2 £ 30 min, and exposed for 1±5 days.
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Acknowledgements We are thankful to Drs T. Lufkin and J. Bieker for providing us with mouse embryonic and adult brain cDNA libraries, respectively. We thank Drs W.-D. Wang, T. Lufkin and members of Stuhlmann's laboratory for thoughtful discussion and suggestions during this work; and Dr T. Lufkin for comments on the manuscript. GenBank accession number for the mouse Sep3 cDNA is AF104411. This work was supported by a Public Health Service First Award no. R29 HD-31534, and a Grant-in-Aid from the American Heart Association, New York City Af®liate to H. S. J.-W. X. was in part supported by NIH training grant 1 T32 DK07757-01. References Byers, B., Goetsch, L., 1976. A highly ordered ring of membrane-associated ®laments in budding yeast. J. Cell Biol. 69, 717±721. Fares, H., Peifer, M., Pringle, J.R., 1995. Localization and possible functions of Drosophila septins. Mol. Biol. Cell 6, 1843±1859. Friedrich, G., Soriano, P., 1991. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Gene Dev. 5, 1513±1523. Gossler, A., Zachgo, J., 1993. Gene and enhancer trap screens in ES cell chimeras. In: Joyner, A.L. (Ed.). Gene Targeting: A Practical Approach, Oxford University Press, New York, pp. 181±227. Gossler, A., Joyner, A.L., Skarnes, W.C., 1989. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244, 463±465. Hartwell, L.H., 1971. Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergenece and cytokinesis. Exp. Cell Res. 69, 265±276. Hogan, B., Beddington, R., Constantini, F., Lacy, F., 1994. Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Hsu, S.-C., Hazuka, C.D., Roth, R., Foletti, D.L., Heuser, J., Scheller, R.H., 1998. Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin ®laments. Neuron 20, 1111±1122. Kato, K., 1990. A collection of cDNA clones with speci®c expression patterns in mouse brain. Eur. J. Neurosci. 2, 704±711. Kato, K., 1992. Finding new genes in the nervous system by cDNA analysis. Trends Neurosci. 15, 319±323. Kinoshita, M., Kumar, S., Mizoguchi, A., Ide, C., Kinoshita, A., Karaguchi,
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T., Hiraoka, Y., Noda, M., 1997. Nedd5, a mammalian septin, is a novel cytoskeletal component interading with actin-based structures. Genes Dev. 11, 1535±1547. Kumar, S., Tomooka, Y., Noda, M., 1992. Identi®cation of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155±1161. Leahy, A., Xiong, J.-W., Kuhnert, F., Stuhlmann, H., 1999. Use of developmental marker genes to de®ne temporal and spatial patterns of differentiation during embryoid body formation. J. Exp. Zool. 284, 67±81. Longtine, M.S., DeMarini, D.J., Valencik, M.L., AI-Awar, O.S., Fares, H., Virgiho, C.D., Pringle, J.R., 1996. The septins: roles in cytokinesis and other processes. Curr. Opin. Cell Biol. 8, 106±119. Lupas, A., 1996. Prediction and analysis of coiled-coil structures. Methods Enzymol. 266, 513±525. McKie, J.M., Sutherland, H.F., Harvey, E., Kim, U.J., Scambler, P.J., 1997. A human gene similar to Drosophila melanogaster peanut maps to the DiGeorge syndrome region of 22q11. Hum. Genet. 101, 6±12. Nagase, T., Seki, N., Ishikawa, K., Ohira, M., Kawarabayasi, Y., Ohara, O., Tanaka, A., Kotani, H., Miyajima, N., Nomura, N., 1996. Prediction of the coding sequences of unidenti®ed human genes. VI. The coding sequences of 80 new genes (KIAA0201-KIAA0280) deduced by analysis of cDNA clones from cell line KG-1 and brain. DNA Res. 3, 321± 329. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J.C., 1993. Derivation of completely cell culture-derived mice from early passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 824±828. Nakatsuru, S., Sudo, K., Nakamura, Y., 1994. Molecular cloning of a novel human cDNA homologous to CDC10 in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 202, 82±87. Neufeld, T.P., Rubin, G.M., 1994. The drosophila peanut gene is required for cytokinesis and encodes a protein similar to yeast putative bud neck ®lament proteins. Cell 77, 371±379. Nottenburg, C., Gallatin, W.M., John, T.S., 1990. Lymphocyte HEV adhesion variants differ I n the expression of multiple gene sequences. Gene 95, 279±284. Saraste, M., Sibbald, P.R., Wittinghofer, A., 1990. The P-loop: a common motif in ATP- and GTP-binding proteins. Trends Biochem. Sci. 15, 430±434. Xiong, J.-W., Battaglino, R., Leahy, A., Stuhlmann, H., 1998. Large-scale screening for developmental genes in ES cells and embryoid bodies using retroviral entrapment vectors. Dev. Dyn. 212, 181±197. Xiong, J.-W., Leahy, A., Lee, H.-H., Stuhlmann, H., 1999. Vezf1: a Zn ®nger transcription factor restricted to endothelial cells and their precursors. Dev. Biol. 206, 123±141. Zieger, B., Hashimoto, Y., Ware, J., 1997. Alternative expression of platelet glycoprotein lb(beta) mRNA from an adjacent 5 0 gene with an imperfect polyadenylation signal. J. Clin. Invest. 99, 520±525.
Gene expression pattern
Retroviral promoter-trap insertion into a novel mammalian septin gene expressed during mouse neuronal development Jing-Wei Xiong, Amy Leahy, Heidi Stuhlmann* Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029, USA Received 22 February 1999; received in revised form 4 May 1999; accepted 10 May 1999
Abstract We have characterized a retroviral promoter-trap insertion into a novel mammalian septin gene, Sep3. Its predicted amino acid sequence shares signi®cant homology to that of Saccharomyces cerevisiae CDC3, CDC10, CDC11, CDC12, the Drosophila genes Pnut, Sep1, Sep2, and the mammalian genes BH5, CDC10, Nedd5, Diff6, and Sep2, which are implicated in cytokinesis and cell polarity. Sep3 encodes a protein of 465 amino acids, and contains an evolutionary conserved ATP/GTP-binding motif, two coiled-coil domains, and a highly hydrophobic domain at the C terminus. Alkaline phosphatase reporter gene expression in transgenic embryos was ®rst detected at E8.5 in the neural fold, and high levels of expression continued throughout embryogenesis in the neural tube and brain. In addition, a low level of transient expression was detected in the somites, gut, and branchial arches of mouse embryos. Overall, reporter gene expression recapitulated Sep3 mRNA expression during mouse embryogenesis. In adults, Sep3 transcripts were only detected in the brain and testis. Zoo blot analysis revealed that Sep3-related sequences exist in several vertebrate species including zebra®sh, frog, chicken, mouse and human. Consistent with the retroviral insertion into the 3 0 UTR of the Sep3 gene, no obvious phenotypes associated with the promoter trap were detected in transgenic embryos or adult mice. In summary, we report the ®rst isolation of a novel full-length Sep3 cDNA and extensive characterization of its expression during mouse embryogenesis and in adult tissues. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Septins; Sep3; Promoter trap; Alkaline phosphatase reporter gene; Genetic screen; Neuronal development; Mouse embryogenesis; ES cells; Embryoid bodies; Transgenic mice
1. Results 1.1. AP expression in transgenic 2-86 embryos is largely restricted to the developing CNS In an approach to identify genes involved in early mouse development, we have performed an entrapment vector screen in ES cells and mouse embryos (Xiong et al., 1998). ES cell clone 2-86 was identi®ed as a candidate clone based on the spatially and temporally restricted expression of the alkaline phosphatase (AP) reporter gene during ES cell differentiation into embryoid bodies (Xiong et al., 1998). To examine AP reporter expression in vivo and to determine if the promoter-trap insertion caused a mutant phenotype, we established a transgenic mouse line by using the diploid embryo-ES cell aggregation technology (Nagy et al., 1993; Xiong et al., 1998). Mice hetero- or homozygous for the 2-86 insertion were viable * Corresponding author. Tel.. 11-212-241-9413; fax: 11-212-8609279. E-mail address: [email protected] (H. Stuhlmann)
and fertile, and did not show overt abnormalities. Heterozygous males carrying the 2-86 insertion were bred with wild-type CD-1 females. Staged embryos were dissected out of their deciduas and subjected to AP staining. The earliest time point of AP expression in whole-mount embryos was detected at E8.5 in a region where folding of the neural plate begins. In E8.5 to E9.0 embryos, strongest AP expression was detected in the anterior neural tube and posterior hindbrain and weaker expression in other regions of the developing CNS, the somites and branchial arches (Xiong et al., 1998). Expression appeared restricted to dorsal aspects of the neural tube that may include neural crest cells, and was not detected in cephalic mesenchyme, foregut, or branchial arches (Fig. 1A,B). In transverse sections through the posterior part of embryo, AP expression was detected in the neural tube, notochord, the dorsal part of the hind gut, and somites (Fig. 1C,D). In wholemount E10.5 embryos, strong AP expression was found throughout the brain, in the optic vesicle, and the spinal cord, and weak expression in the somites and branchial arches (Xiong et al., 1998). Strong AP expression was
0925-4773/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00113-6
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Fig. 1. AP reporter gene expression in 2-86 transgenic embryos. Transgenic embryos were isolated, ®xed and stained for AP, post-®xed, embedded in paraf®n, and sectioned. (A±D) Transverse sections of E8.5 embryo through head region (A), foregut and branchial arches (B), hind gut (C), and posterior trunk (D), respectively. (E,F) Transverse sections of E9.5 embryo through optic vesicle (E), and Rathke's pouch (F). Abbreviations: BRA, branchial arch; CM, cephalic mesenchyme; E, epidermal ectoderm; FG, fore gut; HG, hind gut; NE, neural epithelium; NT, neural tube; OV, optic vesicle; RP, Rathke's pouch; SOM, somites.
detected in transverse sections through the head region, predominantly in the dorsal epithelium of the optic vesicle, and apparently localized to the future lens epithelium, the overlying corneal ectoderm and surrounding mesenchyme (Fig. 1E). Expression was also detected in Rathke's pouch region (Fig. 1F), the brain and spinal cord (not shown). 1.2. Molecular cloning of genomic sequences ¯anking the 286 insertion and Sep3 cDNA To understand the 2-86 insertion on the molecular level, we cloned a 9.4 kb 5 0 EcoRI-EcoRI fragment, and a 2.7 kb
3 0 EcoRI-EcoRI fragment by using the supF complementation approach (Xiong et al., 1998; see also Fig. 2A). A 3.0 kb 5 0 NheI-NheI fragment, and a 0.8 kb 3 0 KpnI-EcoRI fragment, containing mostly ¯anking genomic DNA, were subcloned and used as probes for southern analysis to verify that the genomaic clones corresponded to the 2-86 insertion (Fig. 2A). Both probes detected a 6.7 kb pre-integration fragment in EcoRI-digested, genomic DNA from R1 ES cells, clone 2-86, and another unrelated clone, 2-51, with a promoter-trap insertion. In addition, the 3 0 KpnI-EcoRI probe hybridized to a 3.4 kb fragment in DNA from clone 2-86, and DNA from R1 cells mixed with 10 ng of cloned
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genomic 3 0 EcoRI-EcoRI DNA, but not in DNA from clone 2-51 or R1 cells (Fig. 2A). Reprobing the ®lter with a neospeci®c probe detected the same 3.4 kb genomic fragment (not shown). The 5 0 NheI-NheI probe detected a 9.4 kb fragment in DNA from clone 2-86, but not in DNA from clone 2-51 or R1 cells (Fig. 2A). Since the design of the PT-I RES-AP vector predicted insertion into an exon of a targeted endogenous gene, both ¯anking regions should contain exonic sequences (Gossler and Zachgo, 1993; Xiong et al., 1998). The 0.8 kb 3 0 KpnIEcoRI fragment was used as a probe to screen a day-11.5 mouse total embryo cDNA library (Clonetech), and ®ve independent cDNA clones were isolated, ranging from 0.5 to 2.0 kb in length. Sequencing revealed that they shared signi®cant homology with several members of the septin family. Clone #12-1 was the longest cDNA clone, and its sequence corresponds to nucleotides 470±2455 of the fulllength Sep3 gene. To obtain a full-length cDNA clone, a subclone of this cDNA, #12-1 B (corresponding to nucleotides 470±820), was used as a probe to screen an adult brain cDNA library. Several cDNA clones containing the complete coding regions of Sep3 were isolated. The longest cDNA clones contained 2455 bp of sequence (Fig. 2B). Alignment of the 3 0 ¯anking genomic DNA sequence with that of Sep3 cDNA revealed that the retroviral insertion in clone 2-86 mapped to the 3 0 untranslated region of this gene (underlined, bold nucleotides TG at 1821±1822 in Fig. 2B). Consistent with the 2-86 insertion in the non-coding 3 0 region of the Sep3 gene, no obvious phenotypes were detected in 2-86 transgenic embryos or adult mice (see above).
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al., 1992; Kinoshita et al., 1997), with 99% identity to human NED5 in its amino acid sequence. Genetic and molecular analysis of septins from yeast, Drosophila and mammals revealed that septins are required for cytokinesis (Longtine et al., 1996) as well as for establishment of cell polarity (Hsu et al., 1998). Alignment of the predicted SEP3 amino acid sequences and that of several other septin proteins uncovered that overall sequence identity ranged from 36% for yeast CDC3 to 50% for mouse NEDD5 (Fig. 2C, and not shown). The putative SEP3 protein contains a highly conserved ATP/GTP-binding domain at the N terminus, containing the P-loop sequence GQSGLGKS (Fig. 2B,C) (Saraste et al., 1990; Neufeld and Rubin, 1994) which may be involved in protein assembly or regulation (Longtine et al., 1996). Based on the Kyte±Doolittle analysis in the MacVector program, a highly hydrophobic region of 60 amino acids is located in the carboxyl terminal region (amino acids 360±420). Two putative coiled-coil domains were identi®ed in two regions of SEP3 (amino acids 129±154 and 175±237, respectively; underlined in Fig. 2B), using the World Wide Web server of the Swiss Institute for Experimental Cancer Research for prediction and analysis of coiled-coil structures (Lupas, 1996). Overall homology between SEP3 and other proteins of the septin family are high except for their N- and C-terminal regions; however, homology among other mammalian septins is higher than between any of those and SEP3 (Fig. 2C, and not shown).
1.3. Sep3 is similar to cell cycle control genes
1.4. Sep3 mRNA expression is found in the developing CNS and somites and is essentially identical to AP expression in transgenic embryos
The nucleotide sequence of the 2455 bp Sep3 cDNA revealed a single open reading frame of 1395 bp that encodes a putative polypeptide of 465 amino acids with a predicted molecular mass of 51 kDa (Fig. 2B). Homology searches of current databases revealed that the putative SEP3 protein is a novel mammalian member of the septin family and shares signi®cant homologies to the yeast cell cycle control proteins CDC3, CDC10, CDC11, and CDC12 (Hartwell, 1971), the Drosophila proteins Pnut, Sep1, and Sep2 (Neufeld and Rubin, 1994; Fares et al., 1995), as well as eight human and four mouse septin genes. The human genes are BH5 (GenBank accession #3290200); CDC10 (Nakatsuru et al., 1994); H5 (McKie et al., 1997; Zieger et al., 1997); KIAA0128 (Nagase et al., 1996); NED5 or KIAA0158 (Nagase et al., 1996); a putative septin (GenBank accession #e1167897); a gene related to Drosophila Sep2 (Nagase et al., 1996); and a CDC-related protein (GenBank accession #3283224). The mouse genes are BH5 (Kato, 1990), with 93% identity to human BH5 in its amino acid sequence; CDC10 (GenBank accession #e1250468); Diff6 (Nottenburg et al., 1990); and NEDD5 (Kumar et
RNA in situ hybridization analysis was used to determine the temporal and spatial distribution of Sep3 mRNA during mouse embryogenesis. A sense riboprobe did not show any speci®c signal (Fig. 3b, panel C). The antisense Sep3 probe detected expression primarily in the developing CNS in E9.5±E13.5 embryos. At E10.5, strong signal was detected in the neural epithelium throughout the fore-, mid-, hindbrain, and the neural tube, and transiently in the surface ectoderm (Fig. 3a, panels A,B). Weaker expression was observed in the dorsal root ganglia, the somites, and gut (Fig. 3a, panels A,C, and not shown). No speci®c signal was detected in heart and liver. At E11.5 and E12.5, Sep3 expression was observed primarily in neuronal tissue and dorsal root ganglia (not shown). At E13.5, high signal stayed on in all parts of the brain and the neural tube, and weaker signal was detected in the dorsal root ganglia (Fig. 3b, panels A,B). Highest Sep3 expression was detected in the outer layer of cells of the fore- and midbrain (Fig. 3b, panels A,B). In summary, Sep3 mRNA expression was located primarily in developing CNS and PNS and was essentially identical to AP expression observed in 2-86 transgenic embryos (compare Fig. 1 to Fig. 3).
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1.5. Sep3 transcripts are detected in mouse embryos and in adult brain and testis To determine the size and distribution of Sep3 transcripts, Northern blot analysis was performed on total RNA from staged embryos between E7.5 and E16.5, and from adult organs (Fig. 4). Two major Sep3-speci®c transcripts of about 6.0 kb and 2.4 kb in length were detected at increasing levels in embryos between E8.5 and E13.5, but not in E7.5 embryos (Fig. 4) or ES cell RNA (not shown). The same two transcripts were detected in brain, but not in any other adult mouse tissues examined. The relative abundance of the two transcripts was similar in all sources of RNA. The smaller transcript of about 2.4 kb in length corresponded in size to the 2.45 kb Sep3 cDNA that we isolated. In adult testes, less abundant transcripts of 4.2 kb and approximately 0.8 kb length were detected. 1.6. Sep3-related sequences are conserved in vertebrates `Zoo blot' analysis was performed to determine whether Sep3-related sequences were present in other vertebrate or invertebrate species. Genomic DNA from zebra®sh, Xenopus, Gallus, mouse and human was digested with BamHI, EcoRI, or HindIII, respectively, and subjected to Southern blot analysis. Filters were hybridized at reduced stringency with a 2.45 kb Sep3 cDNA probe and washed at high stringency. One or two strong hybridizing fragments and several fragments with lower intensity were detected in the genomic DNA from mouse, human and chicken (Fig. 5). In DNA from lower vertebrates (Fig. 5) and sea urchin (not shown), between one and four bands with lower intensity were detected. These results suggest that the mouse Sep3 gene belongs to a small family of genes which share related sequences in other vertebrate species. 2. Discussion We report here on the identi®cation and characterization of the endogenous gene targeted by a promoter trap insertion, Sep3. Sep3 belongs to the evolutionary conserved family of septin genes, and its expression during mouse embryogenesis is located primarily in the developing CNS and PNS. Sep3 encodes a putative protein of 465 amino acids that shares about 36% to 50% overall amino acid sequence identity with known septins in S. cerevisiae,
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Drosophila, mouse and human. It shows several characteristic hallmarks of septins including an evolutionary conserved ATPase/GTPase motif and two coiled-coil domains. In addition, Sep3 contains a highly hydrophobic domain at its C terminus that is distinct from other septins. Current understanding of the biological functions of septins largely comes from analysis of the yeast CDC3/10/11/12 and the Drosophila Pnut mutants, all of which have distinct phenotypes in cytokinesis. Although several septin genes with signi®cant overall amino acid sequence homology are found in each species, each individual septin appears to play an important role rather than displaying functional redundancy or compensation in cytokinesis (Neufeld and Rubin, 1994; Longtine et al., 1996). So far, at least eight mammalian septin genes have been identi®ed and partially characterized. Original ts-lethal mutations including mutations in S. cerevisiae septin genes CDC3, CDC10, CDC11, CDC12, and the Drosophila Pnut mutant have shown defects in cytokinesis (Hartwell, 1971; Neufeld and Rubin, 1994). Furthermore, anti-septin antibodies localized septin gene products to the mother-bud neck in the yeast (Longtine et al., 1996), to the cleavage furrows and residual intercellular bridges in the ¯y (Neufeld and Rubin, 1994; Fares et al., 1995), and near the contractile ring and midbodies in the mouse, respectively (Kinoshita et al., 1997). EM studies suggested that the septin-containing structures are closely associated with the plasma membrane (Byers and Goetsch, 1976; Neufeld and Rubin, 1994; Fares et al., 1995; Kinoshita et al., 1997), and they fractionate largely with the crude membrane fraction (Longtine et al., 1996). RNA in situ hybridization analysis revealed that Sep3 expression is predominantly localized to the neural epithelium and to differentiating, post-mitotic neurons. Interestingly, it has been reported that other septins also display strong expression in the nervous system, e.g. Drosophila septins (Neufeld and Rubin, 1994; Fares et al., 1995), mouse Nedd5 and H5 (Kumar et al., 1992; Kinoshita et al., 1997; Kato, 1990, 1992), and four rat septins (KIAA0128, CDC10, NEDD5, and H5) that are associated with a sec6/8 complex (Hsu et al., 1998). What is the function of septins in these non-dividing neurons? It has been proposed that septins are involved in the generation of the leading edge of membrane extension as well as in establishing cell polarity both in yeast and animal cells (Longtine et al., 1996; Hsu et al., 1998). In particular, both the sec6/8
Fig. 2. Molecular cloning of the 2-86 retroviral insertion site and Sep3 cDNA. Proviral and ¯anking cellular sequences were isolated by using the supF complementation method (Xiong et al., 1998). Sep3 cDNA was isolated and sequenced. Sep3 cDNA and its predicted sequence were analyzed by using the MacVector program. (A) Left: schematic map of the 2-86 proviral integration site with the PT-IRES-AP vector ¯anking genomic DNA. The positions of EcoRI (E) restriction enzyme sites used for supF are indicated. A KpnI (K) and two NheI (Nh) sites were used for cloning of a ¯anking 5 0 I-NheI and a 3 0 KpnI-EcoRI genomic fragment. Right: genomic DNA was digested with EcoRI and transferred to nitrocellular membranes. The blot containing 10 mg R1 ES cell DNA mixed 10 pg cloned 3 0 EcoRI-EcoRI fragment, 10 mg DNA each from ES cell clone 2-86, ES cell clone and R1 ES cells were hybridized with a 0.8 kb [a- 32P] dCTP-labeled 3 0 KpnI-EcoRI fragment 3.0 kb [a- 32P]dCTP-#labeled 5 0 NheI-NheI fragment. The position of the 5 0 and 3 0 EcoRI fragments, and the preintegration EcoRI-EcoRI fragments are indicated by arrow heads. (B) Nucleotide and deduced amino acid sequence of the Sep3 cDNA. The ATPase/GTPase domain is indicated by asterisks, and the two coiled-coil domains are underlined. The 2-86 insertion site, located between nucleotides 1821 and 1822, is shown in bold.
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Fig. 2C. Alignment of SEP3 sequence from amino acid 5 to 443 (top) with the respective sequences from mouse H5, CDC10, NEDD5, DIFF6, and human SEP2.
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Fig. 3. RNA in situ hybridization analysis of Sep3 mRNA in E10.5 and E13.5 embryos. Wild-type embryos were isolated, embedded in paraf®n and sectioned. Sections were processed for RNA in situ hybridization, using anti-sense or sense Sep3 (nucleotides 814±2455) riboprobes. (a) Analysis of E10.5 embryos. (A) Dark-®eld view of a parasagittal section of a E10.5 embryo. High-power magni®cation of hind-brain (B) and dorsal root ganglia (C). (b) Analysis of E13.5 embryos. (A) Dark-®eld view of a parasagital section of E13.5 embryo hybridized with anti-sense Sep3 probe. Dark-®eld view of high-power magni®cation of fore-brain hybridized with anti-sense Sep3 probe (B), or sense Sep3 probe (C). Abbreviations: bra, branchial arch; cm, cephalic mesenchyme; drg, dorsal root ganglia; epe, epidermal ectoderm; fb, forebrain; hb, hindbrain; hrt, heart; mb, midbrain; nep, neural epithelium; nt, neural tube; ov, optic vesicle; rp, rathke's pouch; som, somites.
Fig. 4. Expression of Sep3 mRNA in embryos and adult organs. 20 mg each of total RNA was resolved, transferred, and hybridized with a [a- 32P]dCTPlabeled 2.45 kb Sep3 cDNA fragment. After removal of the probe, the ®lters were re-hybridized with a [a- 32P]dCTP-labeled mouse GAPDH plasmid. (A) RNAs from staged CD-1 embryos between E7.5 and E16.5. (B) RNAs from adult organs of CD-1 mice. LIV, liver; SPL, spleen; KID, kidney; GUT, gut; MUS, muscle; LUN, lung; HRT, heart; THY, thymus; BR, brain; OVA, ovary; TES, testis.
Fig. 5. Southern `zoo blot' analysis of genomic DNA from different vertebrate species. Genomic DNA from different species was digested with BamHl (B), EcoRI (R), or HindIII (H), resolved on 0.8% agarose gels, transferred to nitrocellulose membranes, and hybridized to a [a- 32P]dCTP-labeled Sep3 probe. The ®gure is a composite of X-ray ®lms exposed for 1 day (DNA from chicken, mouse, human), and 4 days (DNA from zebra®sh, Xenopus), respectively.
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complex and septin ®laments are implicated in traf®cking vesicles and organizing proteins at the plasma membrane of neurons (Hsu et al., 1998). It will be of interest to determine whether SEP3 is involved in formation of the septin ®lament and sec6/8 complexes in neurons as well. The promoter-trap approach has been successfully applied to `tag' and to inactivate cellular genes in mouse ES cells and embryos (Gossler et al., 1989; Friedrich and Soriano, 1991). In this report, we have identi®ed Sep3 as the endogenous gene targeted by the 2-86 insertion. Consistent with its insertion into the 3 0 UTR of the Sep3 gene, no obvious phenotype was detected in transgenic embryos and adult mice derived from the 2-86 ES cell clone. In the future, loss-of-function analysis of Sep3, together with immunohistochemical localization of SEP3 in the developing CNS, will allow us to dissect its function during early mouse development. 3. Experimental procedures 3.1. Generation of ES cell aggregation chimeras and transgenic mice Clone 2-86 ES cells were used to generate germ-line chimeras by employing the ES cell-diploid embryo aggregation method (Nagy et al., 1993; Xiong et al., 1998). Three highly chimeric males transmitted the provirus through the germline. Inbred mice heterozygous and homozygous for the insertion were generated by crossing F1 founder males with 129/Sv females or b y intercrossing of heterozygous F1 males and females and genotyped by using a 0.8 kb KpnIEcoRI 3 0 ¯anking fragment as a probe (Fig. 2A). 3.2. AP staining of whole-mount embryos and histological sections Staged embryos were dissected from their deciduas of pregnant CD-1 females that were mated with heterozygous 2-86 males. Mouse embryos and extraembryonic membranes from stages E7.5±E13.5 were ®xed, heat-treated for 30 min at 708C, and subjected to AP staining using BM purple BCIP/NBT substrates (Boehringer Mannheim) as described (Xiong et al., 1998). Whole-mount stained embryos were post-®xed, embedded in paraf®n, and sectioned by conventional methods (Hogan et al., 1994). Photographs were taken under a Nikon SMZ-U stereomicroscope, or a Zeiss Axioskop microscope. 3.3. Molecular cloning of genomic DNA ¯anking the retroviral insertion and cDNA Genomic DNA containing retroviral vector, 5 0 and 3 0 ¯anking genomic sequences from clone 2-86 were isolated by supF complementation as described previously (Xiong et al., 1998). A 0.8 kb KpnI-EcoRI fragment containing 109 bp of 3 0 Mo-MLV long terminal repeat (LTR) and about 700 bp
3 0 ¯anking genomic sequences was used as a probe to screen an E11.5 mouse embryonic cDNA library (Clontech). The two longest overlapping clones, #8-3-1, and #12-1, were subcloned and sequenced on both strands by using an automatic DNA sequencer (ABI50, Utah State University). Resulting cDNA sequences lacked most of 5 0 sequences, and corresponded to nucleotides 470±2455 of Sep3 gene. In order to obtain a full-length cDNA clone, the most 5 0 EcoRI-EcoRI fragment of clone #12-1, #12-1B, was used as a probe to screen an adult mouse brain cDNA library (Stratagene). The three longest clones, #3, #11, and #12, were subcloned and sequenced as described above. All three clones contained the full-length coding region of the Sep3 gene. 3.4. RNA in situ hybridization Sep3 cDNA clone #12-1 A (corresponding to nucleotides 814±2455) was used as a template for the synthesis of sense and antisense riboprobes by RNA in vitro transcription. In situ hybridization to paraf®n-embedded embryo sections was performed as described previously (Leahy et al., 1999) 3.5. RNA isolation and Northern hybridization analysis Total RNA was prepared from staged embryos and adult mouse tissues by using a modi®cation of the guanidiniumCsCl method as described (Xiong et al., 1999). 20 mg samples of RNA were electrophoresed on 1% agaroseformaldehyde gels and transferred in 20 £ SSC to nitrocellulose membranes (Optitran, Schleicher and Schuell). Filters were hybridized at 428C in 50% formamide with 1 £ 107 cpm of a random hexamer-primed, [a- 32P]dCTPlabeled 2.45 kb fragment containing the full-length Sep3 cDNA. Filters were washed at 508C in 0:2 £ SSPE (36 mM NaCl; 2 mM NaPO4, pH 7.7; 0.2 mM EDTA), 0.1% SDS and exposed for 1±2 days. To control for RNA loading, ®lters were stripped and hybridized with a [a- 32P]dCTPlabeled mouse GAPDH cDNA plasmid. 3.6. Southern analysis of genomic DNA and `zoo blot' analysis Preparation of high molecular weight DNA, digestion with restriction endonucleases, and southern analysis was performed as described previously (Xiong et al., 1999). For analysis of the 2-86 integration site, ®lters were hybridized with a 0.8 kb 3 0 KpnI-EcoRI fragment, or a 3.0 kb 5 0 NheINheI fragment, respectively. For `zoo blot' analysis, ®lters were hybridized to 2 £ 107 cpm of a [a- 32P]dCTP-labeled 2.45 kb full-length Sep3 cDNA under reduced stringency conditions (358C in 50% formamide, 5 £ SSC, 2 £ Denhardt's, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA). Blots were washed at increasing stringency, with the ®nal washes at 658C in 0.2% £ SSC, 0.1% SDS for 2 £ 30 min, and exposed for 1±5 days.
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