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The Drosophila Homologue of Vertebrate Myogenic-Determination

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The Drosophila Homologue of Vertebrate Myogenic-Determination Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3782-3786, May 1991 Developmental Biology The Drosophila homologue of vertebrate myogenic-determination genes encodes a transiently expressed nuclear protein marking primary myogenic cells (insect myogenesis/helix-loop-helix/invertebrate MyoD) BRUCE M. PATERSON*t, UWE WALLDORFt, JUANITA ELDRIDGE*, ANDREAS DUBENDORFERt, MANFRED FRASCH§, AND WALTER J. GEHRINGt *Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; tDepartment of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland; tZoology Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; and §Max Planck Institute for Developmental Biology, Abteilung III Genetik, D-7400 Tubingen, Federal Republic of Germany Contributed by Walter J. Gehring, January 22, 1991 10 30 50 ABSTRACT We have isolated a cDNA clone, called Dmyd GGAAAAATCCCGAAAGTGAAACTATAACAAATTAAACTAAATAGAAACCACAGCCTAAAA 70 90 110 that encodes a CTTGTGTGTAACCATAACTCATAAATTTGTGTTAATGACCTAGCATACCTAGAAAAGGAG for Drosophila myogenic-determination gene, 130 150 170 CTTAAACCACGTTAAAAATGGTAATAATCAACTGACTAAATTAACTGTGCCATCGTAATT protein with structural and functional characteristics similar to 190 210 230 AGTATCAGCAATTAAATTCAGCAACTTGTTGAACTACCAAAATTGTTTTGTAAAATATAA the members of the vertebrate MyoD family. Dmyd clone 250 270 290 CAAAATCGTAGTGAAGTGAAAAATGACCAAGTATAkATAGTGGCAGCAGTGAAATGCCTGC encodes a polypeptide of 332 amino acids with 82% identity to M TK Y N S G S S E M P A 310 330 350 MyoD in the 41 amino acids of the putative helix-loop-helix GGCTCAAACCATCAAGCAGGAGTACCACAATGGCTATGGTCAGCCGACACATCCTGGATA A Q T I K 0 E Y H N G YG Q P T H P G Y region and 100% identity in the 13 amino acids of the basic 37S0 390 4 10 CGGATTTAGCGCCTATAGCCAACAGAATCCGATAGCCCATCCCGGCCAGAATCCACACCA domain proposed to contain the essential recognition code for G FS A Y S QQ N P I A H P G Q N P H Q 430 450 470 muscle-specific gene activation. Low-stringency hybridizations GACACCTGCAGAATTTCTTTAGCCGCTTCAATGC CCGGGTGATGCGAGTGCGGGAAATGG T L Q N F F S R F N A V G D A S A GN G indicate that Dmyd is not a member of a multigene family 4 90 510 530 TGGAGCGGCTTCCATCTCAGCCAACGGATCGGGTTCGTC TTGCAACTACAGTCATGCGAA to in vertebrates. is a nuclear in G A A S I S A NG S G S S C N Y S H A N similar MyoD Dmyd protein 550 570 590 TCATCATCCGGCGGAGCGTGGACAAGCCGTTGGGCATGAATATGACAC CGTCGCCCATCTA Drosophila, consistent with its role as a nuclear-gene regulatory HH P A E L D K P L G M N M TPSP I Y 610 630 650 factor, and is proposed to be a transiently expressed marker for CACCACGCGACTAGATGACGAGAACAGCAGTCTCAGCTCCGAGGAGCACGTCCATGCGCC T TD Y DD E N S S L S S E E H V H A P muscle founder cells. We have used an 8-kilobase promoter 670 690 710 CCTCGTCTGCTCCTCCGCCCAATCCTCCAGACCATGCCTCACCTGGGCCTGCAAGGCGTG fragment from the gene, which contains the first 55 amino acids L V C SS A Q SS R P CL T W A C K A C 730 750 770 of the Dmyd protein, joined to lacZ, to follow myogenic CAAAAAGAAGAGCGTCACCGTGGACCGTCGAAA.AGCGGCCACTATGAGGGAACGCCGGAG JK K K ,IS V T V D 0A A T MRL EPR R R precursor cells into muscle fibers with antibodies to 13-galac- 1 BAS IC 2 3 790 910 830 tosidase and to Dmyd. Unlike the myogenic factors in verte- TGCCGAAAGGTTAACTGAGGCCTTCGAGATCTCTTCAAGCGACGCACTTCAATAATCOCCAA NLR K V NE A F E IOL K R0TR FS N P N brate muscle cells, Dmyd appears to be expressed at a much HELIX 1 850 870 890 lower level in differentiated Drosophila muscles, so Dmyd CCAGCGCCTGCCGAAGGTTGAGATATTGCGCAATGCCATCGAG TATATCGAGAGCCTGGA Q R L P K V 'E|I L R N A I E Y I ES L E cannot be followed as a muscle marker. This fact LOOP HELIX 2 continuously 910 930 950 RNA in 12- to 24-hr GGATCTGCTACAGGAATCCAGTACCACACGCGATGGCGACAACCTGGCGCCCAGTTTGAG is reflected in the loss of Dmyd expression JD L L Q E S S T T R D G D N L A P S L S as well as in the 9170 990 1010 embryos, a major period of early myogenesis, CGGCAAAAGCTGCCAGTCCGATTATCTGAGCTCCTATGCTGGCGCTTATCTAGAAGATAA G K S C Q S D YL SS Y A G A Y LED K undetectable level of the nuclear antigen in primary cultures of 1030 1050 1070 ACTTAGTTTTTACAACAAACATATGGAGAAlTATGGTCAGTTTACAGACTTTGATGGCAA embryonic and adult Drosophila muscle. L SF Y N K H M E K Y G Q FT D FD GN 1090 1110 1130 TGCCAATGGCTCCAGTTTGGACTGTCTAAATCTGATTGTTCAGAGCATCAATAAGAGCAC A N G S S L D C L N L I V Q S I N K S T The discovery of the four myogenic-determination genes 1150 1170 1190 CACGAGTCCCATTCAAAATAAGGCCACGCCCTCCGCTTCAGATACCCAATCGCCGCCCTC (1-4) in the vertebrates has helped to establish the idea that T S P I Q N K A T P S A S D T Q SP P S 1210 1230 1250 a small number of so-called "master regulatory genes" select ATCCGGAGCAACTGCACCCACTTCTCTGCACGTGAACTTCAAACGGAAGTGCAGCACTTA S G A T A P T S L H V N F K R K C S T* and commit embryonic cells to a particular developmental 1270 1290 1310 GCACTTAAGTATCAGCACCTTAGGCAATTGTAAAGCTATTTTTAAGAGGATACACGAGAT pathway. All members of this gene from to 1330 1350 1370 family, amphibia ACCCAGTGACCCGAATAGGCCTTAAATTATTTGTATAGCATTAGAACTTAATTAAATGGT man, share a common structural motif, a basic domainjoined 1390 1410 to a putative helix-loop-helix configuration, that is essential AATTCAAAACAGCAAAAAAAAA for function (5). FIG. 1. Nucleotide and predicted amino acid sequence of the To analyze early developmental events leading to muscle longest open reading frame in the Drosophila Dmyd cDNA clones. formation (6) we have used a probe from the conserved basic The encoded polypeptide has 332 amino acids and a molecular mass helix-loop-helix domain of the avian myogenic factor CMD1 of 36,208 Da. cDNA clone 1 includes nucleotides 1-1313, and clone (7) to isolate a Drosophila cDNA clone, Dmyd, encoding a 2 includes nucleotides 1314-1410. polypeptide very similar to the vertebrate myogenic- determination genes. Here we demonstrate that the Dmyd MATERIALS AND METHODS gene product is a transiently expressed nuclear protein that serves as a marker for myogenic precursor cells, or a subset General Methods. Preparation ofprobes, Drosophila DNA, thereof, in Drosophila development. Similar to the grasshop- RNA, the screening of libraries, Northern (RNA) and South- per (8), these cells may represent the muscle founder cells ern blot analysis, and dideoxynucleotide sequence determi- that are crucial in organizing and establishing the precise nation were done as described (9). muscle pattern in each segment of the Drosophila body plan. Preparation of Primary Cultures of Drosophila Embryo Cells. Single-drop cultures were prepared and treated with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: MHC, myosin heavy chain; EFla, elongation factor in accordance with 18 U.S.C. §1734 solely to indicate this fact. la. 3782 Downloaded by guest on September 29, 2021 Developmental Biology: Paterson et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3783 1 61 Omyf 6. Frg KRKSaptDRR KAATLRERRR LKKINEAFEA LKRrTvaNPN QRLPKVEILR SAIsYIERLQ d transformant lines were generated and balanced. The Dmyd- Mrf4.Frg KRKSaptDRR KAATLRERRR LKCINEAFEA LKRrTvaNPN QRLPKVEILR SAInYIERLQ d Herc. Frg KRKSaptDRR KAATLRERRR LKKINEAFEA LKRrTvaNPN QRLPKVEILR SAIsYIERLQ d lacZ transformant 14.1(11) used in these experiments mapped Hmyf 5. Frg KRKSttmDRR KAATMRERRR LKKVNqAFEt LKRcTttNPN QRLPKVEILR nAIRYIEsLQ e Hmgn. Frg KRKSvsVDRR rAATLkEkRR LKKVNEAFEI LKRsT11NPN QRLPKVEILR SAIqYIERLQ a to 47A. Mmgn.Frg KRKSvsVDRR rAATLREkRR LKKVNEAFEA LKRsTllNPN QRLPKVEILR SAIqYIERLQ a Chkmgn Frg KRICtvsIDRR rAATvREkRR LKKVNEAFEA LKRsTllNPN QRLPKVEILR SAIqYIERLQ s Preparation of Dmyd Antibody. A Rsa I fragment from the Mmd. Frg KRKttnaDRR KAATMRERRR LsKVNEAFEt LKRcTSsNPN QRLPKVEILR nAIRYIEgLQ a Cmd. Frg KRKttnaDRR KAATMRERRR LsKVNEAFEt LKRcTStNPN QRLPKVEILR nAIRYIEsLO a Dmyd cDNA (nucleotides 323-922) was inserted into the Xmd. Frg KRKttnaDRR KAATMRERRR LsKVNEAFEt LKRyTStNPN QRLPKVEILR nAIRYIEsLO a Sma I the transferase fusion vector, Dmd. Frg KkKSvtVDRR KAATMRERRR LrKVNEAFEi LKRrTSsNPN QRLPKVEILR nAleYIEsLe d site of glutathione Consensus EMS--VDA&5AT-REA LKKVNEAFEA LKR-TS-NPN QRLPKVEILR SAIRYIERLQ - 1 2 T-E9 1EF expressed in E. coli JM109, and purified as described (16). BASIC HELIX 1 LOOP HELIX 2 New Zealand White rabbits were injected s.c. with 200-300 ,ug of fusion protein in complete Freund's adjuvant and FIG. 2. Dmyd (Dmd) is closely related to the MyoD. The simi- boosted four times with the same amount of protein in larity of the helix-loop-helix sequences and the invariance in the amino acid sequence of the 13 amino acids that include the basic 2 incomplete adjuvant. Antibody was affinity purified (17). and 3 regions place Dmyd in the MyoD group. Sources for protein Cytochemical Methods. Antibody stainings ofwhole-mount sequences are as follows: Myf6 (Hmyf6) (21), Mrf4 (4), herculin embryos (11), ,B-galactosidase activity stainings (15, 18), and (Herc) (22), Myf5 (Hmyf5) (3), Myf4 (Hmgn) (23), myogenin (Mmgn) chromosomal mapping (19) were done as described. (2), chicken myogenin (Chkmgn) (B.M.P., unpublished work), mouse MyoD (Mmd) (1), chicken MyoD (Cmd) (7), and Xenopus MyoD (Xmd) (24, 25). RESULTS Isolation of Dmyd, a Drosophia Homologue to the Verte- ecdysone as described (10). Cultures were fixed and treated brate Myogenic Determination Genes. By using hybridization for antibody staining as done for the whole-mount embryos conditions of reduced stringency, two cDNA clones were (11). plaque-purified from -5 x 105 recombinant phages; clone 1 Isolation of the Dmyd cDNA and Genomic Clones. The from a A gtll random-primed library (0-16 hr) and clone 2 probe used to isolate the Drosophila Dmyd cDNA clones was from a A gtlO library (3-12 hr). The combined 1410-nucleotide prepared by PCR amplification (12) of the basic helix-loop- sequence of the two clones, called Dmyd for Drosophila helix domain in the avian CMD1 cDNA (7) (from nucleotides 413-637) by using the following primers: 5' primer, Poly Al RNA TGGGCGTGCAAGCATATGAAGAGGAAGACC, and 3' C\J primer, TGCATCCTCCTGGAATTCTTACAGGGCCTG- co 0xCe)) CAG.
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