Isolation and Characterization of Mammalian Homologs of the Drosophila Gene Glial Cells Missing
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12364–12369, October 1998 Developmental Biology Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing JAESANG KIM*†,BRADLEY W. JONES†‡§,CHRISTIANE ZOCK*, ZHOUFENG CHEN*, HAI WANG*, COREY S. GOODMAN§, AND DAVID J. ANDERSON*¶ *Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, CA 91125; ‡Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016; and §Howard Hughes Medical Institute, Division of Neurobiology, Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720 Contributed by Corey S. Goodman, August 14, 1998 ABSTRACT The glial cells missing (gcm) gene in Drosoph- hybridization and reverse transcription–PCR (RT-PCR). The ila encodes a transcription factor that determines the choice results indicate that the major sites of expression in embryos between glial and neuronal fates. We report here the isolation are not neural for either of the rodent genes, although their of two mammalian gcm homologs, Gcm1 and Gcm2, and the expression is highly specific to certain nonneural tissues. Thus, characterization of their expression patterns during embry- their function is, for the most part, not conserved from that of onic development. Although Gcm2 is expressed in neural Drosophila gcm. Interestingly, one of the homologs, Gcm1 but tissues at a low level, the major sites of expression for both of not Gcm2, was able to generate extra glial cells when expressed the mammalian genes are nonneural, suggesting that the in Drosophila and could partially rescue the loss-of-function functions of the mammalian homologs have diverged and phenotype, indicating that Gcm1 shares conserved regulatory diversified. However, when expressed ectopically, Gcm1 can capabilities with gcm. substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical MATERIALS AND METHODS properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the Isolation of Rodent Homologs of gcm. A pair of degenerate 9 T T evolution of the Gcm gene family. oligonucleotide primers (5 -CGGATCCAGACC /CGCCAT / T 9 CTG /CGACAAG-3 corresponding to the sequence coding 9 T Drosophila glial cells missing gcm for amino acids RPAICDK and 5 -CGGAATTCTTC / The gene ( ) controls the T T A A 9 GGGT /CT /GGG /GTGATC /GTG-3 corresponding to the binary fate decision between neuronal and glial lineages (1–3). R Loss-of-function mutations in gcm result in conversion of complementary sequence coding for amino acids HDHP / presumptive glial cells into neurons, whereas ectopic expres- KPE) was used to screen by PCR amplification a rat placental sion of gcm generates additional glial cells at the expense of cDNA library that was built in the plasmid vector pcDNA3 neurons. Based on its nuclear localization and sequence- (Invitrogen) and divided into 50 groups. A positive pool was transformed into Escherichia coli, and the resulting transfor- specific DNA-binding activity, GCM was proposed to be a 32 transcriptional activator for glial-specific genes. Consistent mants were screened with a P-labeled probe derived from the PCR product. Several positive clones were sequenced and with this, multiple copies of the proposed GCM-binding identified as rat Gcm1 (rGcm1). The 39 end of rGcm1 was elements are found in the putative upstream regulatory region determined by 39 rapid amplification of cDNA ends by using of a glial-specific gene, reverse polarity (repo) (4). GCM was a cDNA preparation from embryonic day 14.5 (E14.5) rat also shown to be able to activate a reporter gene in a placental tissue primed with an anchored oligo(dT) primer GCM-binding-site-dependent manner in transiently trans- (59-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCA- fected cells (5). AGCTTTTTTTTTTTTTT-39) as the template. The primers Many vertebrate homologs of Drosophila transcription fac- used for the first 30 PCR cycles were 59-CCTGTGGATT- tors involved in neurogenesis have been isolated and shown to TCAGCAGC-39 and 59-CCAGTGAGCAGAGTGACG-39. be expressed specifically in neural tissues (6, 7). This is Primers for the second 30 cycles were 59-TCGCTTACG- particularly well established for proteins belonging to the basic GCTCTCATC-39 and 59-GAGGACTCGAGCTCAAGC-39. helix–loop–helix family (8). However, no mammalian genes The same 32P-labeled probe was used to screen a 129SvEv thus far discovered fulfill the role of a glial fate-determination mouse genomic DNA library built in the phage vector l DASH gene. Thus, it was of significant interest to examine the possible II (Stratagene). The positive clones were divided into two role of mammalian homologs of the gcm gene in the neuron– groups based on the strength of hybridization. Partial sequence glia fate decision. Recently, two mammalian gcm homologs analyses confirmed that the strongly hybridizing group con- have been identified (4, 9). Not surprisingly, the DNA-binding tained Gcm1 clones, whereas the other was composed of a domain, now called the gcm motif, is conserved, whereas the distinct yet homologous gene called Gcm2. Several overlap- rest, likely responsible for interaction with other proteins, is ping genomic clones were sequenced, and from over 5 kb of not. Although one report made a preliminary claim that one composite sequence, a partial cDNA sequence for mouse of the homologs is expressed in embryonic neural tissues (4), Gcm2 (mGcm2) was proposed (see Results). no detailed study of the expression pattern has been presented thus far. Abbreviations: CNS, central nervous system; En, embryonic day n; We describe here the isolation of the two rodent Gcm genes GCM, glial cells missing; mGCM, mouse GCM; PTH, parathyroid and the characterization of their expression patterns by in situ hormone; REPO, reverse polarity; rGCM, rat GCM; RT-PCR, reverse transcription–PCR; UAS, upstream activating sequence. The publication costs of this article were defrayed in part by page charge Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF081556, payment. This article must therefore be hereby marked ‘‘advertisement’’ in AF081557, and AF081558). accordance with 18 U.S.C. §1734 solely to indicate this fact. †J.K. and B.W.J. contributed equally to this work. © 1998 by The National Academy of Sciences 0027-8424y98y9512364-6$2.00y0 ¶To whom reprint requests should be addressed. e-mail: mancusog@ PNAS is available online at www.pnas.org. cco.caltech.edu. 12364 Developmental Biology: Kim et al. Proc. Natl. Acad. Sci. USA 95 (1998) 12365 To isolate a full-length mGcm2 cDNA, two successive described (2). The sca-Gal4 line was obtained from C. Kla¨mbt 30-cycle rounds of PCR amplification were run with a cDNA (13). preparation from E13.5 mouse pharyngeal tissue as the tem- Generation and Analysis of UAS-Gcm Transgenic Reporter plate. The oligonucleotide primers used were based on the Lines in Drosophila. A 1.4-kb rGcm1 cDNA was subcloned as genomic DNA sequence and the subsequently published an EcoRI fragment into the EcoRI site of pUAST, a UAS mGcm2 cDNA sequence (GenBank accession no. D88611; ref. reporter P-element vector (14). The mGcm2 cDNA (from 4). For the first 30-cycle round, primers were 59-CTCTT- p5G2–24) and the mGcm2-myc cDNA (from p5G2-myc) were TCAGGGCCCTGACTAG-39 and 59-CATAAATGCAC- first subcloned as SacI–XbaI fragments into pUC18, then CCTTGGCGTG-39; for the second 30 cycles, primers were reisolated as EcoRI–XbaI fragments, and subcloned into the 59-GATCGAATTCAATGCCAGCAGACAGCACG-39 and EcoRI and XbaI sites of pUAST. Transgenic lines were 59-GATCGGATCCCTGGATTTCTCTTAAAAGTCC-39. generated by P-element-mediated transformation by standard The resulting full-length product was ligated to pCR2.1 with a procedures. Panneural expression was achieved by crossing TA Cloning kit (Invitrogen; now called pG2–24). To generate these lines with sca-Gal4. Expression of UAS-gcm and UAS- a mGcm2 construct containing the 59 UTR (untranslated rGcm1 in a gcm null background was achieved by first recom- region), a pair of primers (59-GATCGAGCTCCTTTGTGT- bining second-chromosome inserts with a gcmDP1 second chro- GTATATCTGCCTC-39 and 59-TGTCCCACGTGAGTT- mosome to create the following stocks: w; P[w1 UAS-gcm]2 TCATCC-39) was used to PCR amplify from genomic DNA a gcmDP1/CyO and w; P[w1 UAS-rGcm1]2B gcmDP1/CyO. These 300-bp product containing 220 bases of 59 UTR and parts of lines were crossed against w; gcmDP1 sca-Gal4/CyO. the first exon. This product was digested with SacI and PmlI Immunohistochemical Detection of REPO Protein in Dro- and inserted into pG2–24 (p5G2–24). To generate a construct sophila Embryos. Recombinant REPO-fusion protein was with a MYC-epitope tag, a three-way ligation was performed produced in E. coli with the QIAexpress system (Qiagen, with the following DNA fragments: (i) p5G2–24 digested with Chatsworth, CA). A 1.4-kb BamHI-HindIII fragment from the BsgI and XhoI, (ii) a PCR product amplified from p5G2–24 repo cDNA pcrepo-2.6 (15) encoding for amino acids 219–612 with primers 59-TCGAGTCCATATTCCACCCTG-39 and 59- was cloned into the pQE-30 expression vector. The resulting GATCGGATCCAAAGTCCTCATTGTCAAAGC-39 di- fusion protein has a 22-aa N-terminal addition that contains six gested with BsgI and BamHI, and (iii) a PCR product amplified histidine residues, allowing for a one-step purification by from pCS21MT (10) with primers 59-TGCAGGATCCCATC- immobilized Ni21 chelate-affinity chromatography. Mice were GATTTAAAGC-39 and 59-GCATCTCGAGTTAGGTGAG- injected with 50 mg of protein emulsified in RIBI adjuvant GTCGCCCAAGCTCTC-39 digested with BamHI and XhoI. (Immunochem Research, Hamilton, MO) and were boosted at The resulting construct has the 59 UTR, mGcm2 ORF, and five 2-week intervals. Horseradish peroxidase immunohistochem- MYC-epitope tags in tandem (p5G2-myc). istry and embryo dissections were carried out as described In Situ Hybridization.