Two Human Homeobox Genes, Cl and C8

Home , Maternal effect, Morphogenesis, Ontogeny

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 4914-4918, July 1987 Developmental Biology Two human homeobox genes, cl and c8: Structure analysis and expression in embryonic development ANTONIO SIMEONE*, FULVIO MAVILIOt, DARIO ACAMPORA*, ADELE GIAMPAOLOt, ANTONIO FAIELLA*, VINCENZO ZAPPAVIGNAt, MAURIZIo D'EsPosITO*, MARIA PANNESE*, GIOVANNI Russot, EDOARDO BONCINELLI*, AND CESARE PESCHLEt§ *International Institute of Genetics and Biophysics, Via Marconi 10, 80125 Naples, Italy; tDepartment of Hematology, Istituto Superiore di Saniti, Viale Regina Elena 299, 00161 Rome, Italy; and tDivision of Obstetrics and Gynecology, Ospedale di Avellino, 83100 Avellino, Italy Communicated by Frank H. Ruddle, April 14, 1987 (received for review February 18, 1987)

ABSTRACT Two human cDNA clones (HHO.cl.95 and scripts, respectively, which are differentially expressed in a HHO.c8.5111) containing a homeobox region have been charac- variety of organs or body parts at 5 to 9 weeks of embryon- terized, and the respective genomic regions have been partially ic/fetal development. analyzed. Expression of the corresponding genes, termed cl and c8, was evaluated in different organs and body parts during METHODS human embryonic/fetal development. HHO.cl.95 apparently Human Embryos and Fetuses. Human embryos and early encodes a 217-amino acid protein containing a class I fetuses were obtained virtually intact from legal curettage homeodomain that shares 60 out of 61 amino acid residues with abortions and were maintained in sterile culture medium at the Antennapedia homeodomain of Drosophila melanogaster. +40C until processing (26). The age of embryos (5-8 weeks) HHO.c8.5111 encodes a 153-amino acid protein containing a was carefully established by morphologic staging according homeodomain identical to that of the frog ACI gene. Clones to multiple criteria (dating error was ± 2 days) (27, 28). Early HHO.cl and HHO.c8 detect by blot-hybridization one and two fetuses (9 weeks) were staged on the basis of standard specific polyadenylylated transcripts, respectively. These are age/crown-rump length plots. Different organs or body parts differentially expressed in spinal cord, backbone rudiments, limb were dissected under an inverted microscope and were stored buds (or limbs), heart, and skin of human embros and early in liquid nitrogen until analyzed. fetuses in the 5- to 9-week postfertilization period, thus suggesting cDNA Clones. Recombinant clones (8 x 105) of a cDNA that the cl and c8 genes play a key role in a variety of library prepared from poly(A)+ RNA of SV40-transformed developmental processes. Together, the results of the human fibroblasts (29) were originally screened with a 600-bp embryonic/fetal expression of cl and c8 and those of two BamHI-Pvu II fragment of clone 903 (4, 30) containing the previously analyzed genes (cdO and c13) indicate a coherent entire Antp homeobox region or a 1050-bp Pvu II fragment pattern of expression of these genes in early human ontogeny. containing the homeobox and the 3' region of theftz gene (4), both labeled to a high specific activity by nick-translation In the fruitfly Drosophila melanogaster, studies on develop- (31). Clones (2.5 x 105) of the same cDNA library were mental mutants have allowed the identification of a number subsequently screened with the insert of clone HHO.cl or of genes controlling morphogenesis, which are classified in HHO.c8 under stringent conditions. Recombinant positive three general groups: (i) maternal-effect genes, which specify clones were isolated, and the longest inserts (HHO.cl.95, the structure and spatial coordinates of the egg, (it) segmen- HHO.c8.5111) were characterized by restriction endonucle- tation genes, which determine the number and polarity of ase mapping and were sequenced (32). body segments, and (iii) homeotic genes, which specify the RNA and DNA Analysis. Total RNA was extracted from identity of each segment (reviewed in ref. 1). Most homeotic fresh or frozen samples by the guanidinium isothiocyanate genes identified so far are clustered in the Antennapedia technique and was selected for poly(A)+ RNA by one passage (Antp-C) and the bithorax (bx-C) complex (2, 3). Cloning of on an oligo(dT)-ceilulose column (33, 34). Ten micrograms of some of these genes has led to the identification of a total RNA or 2-3 pug of poly(A)+ RNA was electrophoresed 183-base-pair (bp) sequence, termed the homeobox, which is on a 1.0% agarose/formaldehyde gel, blotted on nitrocellu- contained in most segmentation and homeotic genes and lose or nylon membranes, and hybridized to 107 cpm ofDNA encodes a highly conserved protein domain (4, 5). Homeo- probe labeled by nick-translation to a specific activity of 3 X box-containing genes have also been cloned from sea urchin 108 to 8 x 108 dpm per Ag (31). The blots were washed under (6), frog (7-9), mouse (10-20), and human (21, 22); however, stringent conditions (cf. ref. 25). Probes were then removed direct evidence for their regulatory role in development is still by boiling in a 0.1% NaDodSO4 buffered solution, and the unavailable (cf. ref. 23). filters were rehybridized to a chicken .8-actin probe for We have reported the isolation of human cDNA clones, normalization (see refs. 24 and 25). Southern analysis was termed HHO.cl, HHO.c8, HHO.c1O, and HHO.c13, con- performed according to standard techniques (31). taining homeobox sequences that appear to represent transcripts from four different genes (22). HHO.c1O (24) and RESULTS AND DISCUSSION HHO.c13 (25) contain a conserved class I homeobox and The nucleotide sequence of HHO.cl.95 is shown in Fig. lA. detect by blot hybridization specific transcripts during human Conceptual translation of its longest open reading frame, embryogenesis. We report here the characterization ofclones starting from the ATG codon at position 100, produces a HHO.cl.95 and HHO.c8.5111 and part of the corresponding peptide sequence of 217-amino acid residues that contains a genomic regions. HHO.cl and HHO.c8 detect by blot hy- class I homeodomain. The carboxyl-terminus of this protein bridization one and two specific polyadenylylated tran- lies 20-amino acid residues downstream from the homeodomain and ends in a row of six glutamic acid residues. A 609-bp The publication costs of this article were defrayed in part by page charge 3'-untranslated region contains a polyadenylylation signal payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed.

4914 Developmental Biology: Simeone et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4915

(AATAAA) 20 nucleotides upstream from the poly(A) tail. A HHO.c1.95 contains a second open reading frame, which putative glycosylation site (Asn-Xaa-Thr) is present at the might ehcode a 156-amino acid protein, starting from the 60th amino acid residue of the homeodomain (Fig. LA), as is methionine codon at position 116 through a TGA codon at found in the Antp homeodomain (23). The original clone position 584 (halfway through the homeobox). Further anal- HHO.cl is identical to HHO.c1.95 at positions 450-1359.

A TTAMCCCCCAGCACGATGCACGACCAGGGCTTGACCTTGCCAGCTGCACATGCCTCCGCCCGTCCTCTCTCCCA 75 A CGTTTTCGTCTAAATCTGC.ACTCTAATTCTGTAATATATCAAGCAATCTCCTAAA 55 GCCTCTTCCTCCTGCCCCCTTCTTCCAMCTCTCCTTAATTTTCTTTCCCTTTTCCATCATTATMTTATTTTTATTTTT 156 Met Ser Ser Leu Tyr Tyr Ala Aan Ala ACCGACACTMAACGTCCCTGCCTACAAATCATCCGCCCAAATTATG ACT TCA TTC TAT TAT (CG AAT GCT 126 A GMTTTATATAMACTATATCTGTCGTCGTCTCCASCTCAGACAGCTCCCCAGCCGCACAGMTCACSCAACGACCACM 233 10 20 Leu Phe Ser Lys Tyr Pro Ala Ser Ser Ser Val Phe Ala Thr Cly Ala Phe Pro Glu GIn -7- TTA TTT TCT MA TAT CCA CCC TCA ACT TCG CTT TTC GCT ACC CGA GCC TTC CCA GAA CAA 186 30 40 Thr Ser Cys Ala Phe Ala Ser Asn Pro Gln Arg Pro Gly Tyr Gly Ala Gly Ser Gly Ala TCMTTCCACCCCCTATCATCCACTCAGGCATTTCTCCACCTATGCACCGCCCGTCCCCCAaAACCC(CATCTACTCCAC 391 ACT TCT TGT GCC TTT GCT TCC MC CCC CAG CCC CCG GCC TAT CGA GCG CGT TCG GGC GCT 246 A 50 60 TCCCTTTTATTCCCCACACCACMTGTCCTGTTCAGTTCCACCCGCGCCCCCTATCACTATGCATCTAATTCCTTTTAC 470 Ser Phe Ala Ala Ser cet Gln Gly Leo Tyr Pro Gly Gly Gly Gly Met Ala Cly Gln Ser A TCC TTC GCC GCC TCG ATG CAG GGC TTC TAC CCC CGC GCC CGG GGC ATG GCG CAG ACC 10 GCC 306 Cys Arg Thr His Thr Thr A Met Leu Ser Asn Gln Acn Leu Cly Asn Gln Ser 70 80 CAGCACGAAGACATG CTC TCA MC TCC ACA CAA AAC ACC TTA SCA CAT MC ACA CAC ACC TCA 533 Ala Ala Gly Val Tyr Ala Ala Gly Tyr Gly Leu Glu Pro Ser Ser Phe Asn Met His Cys GCG GCC GGC GTC TAC GCC CCC CCC TAT GGG CTC GAG CCC ACT TCC TIC MC ATG CAC TCC 366 20 30 Ile Ala Gln Asp Phe Ser Ser Glu GSn Gly Arg Thr Ala Pro Gln Asp Gln Lys Ala Ser 90 100 ATC GCT CAG GAT TTT ACT TCT GAG CAG GGC AGG ACT GCG CCC CAG GAC CAG AM GCC AGT 593 Ala Pro Phe clu Gln Asn Leu Ser Sly Val Cys Pro Sly Ser Ala Lys Ala Ala Sly Acp 7 GCG CCC TmT GAG CAG MC CTC TCC GGG GTG TGT CCC GCC SAC TCC GCC MG GCG GCG GCC 426 40 S0 Ile Gln Ile Tyr Pro Trp Met Gln Arg Met Asn Ser His Ser Gly Val Gly Tyr Gly Ala 110 120 ATC CAG ATT TAC CCC TCC ATG CAC CGA ATG MT TCG CAC AGT GGG CTC GGC TAC GSA GCC 653 Ala Lys Glu Sln Arg Asp Ser Asp Leoi Ala Ala Clu Ser Acn Ile Tyr Pro Trp Phe Arg 60 70 GCC MS GAG CAG AGG GAC TCG SAC TTC GCG CCC GAG ACT MC TTC CGC ATC TAC CCC TCG 486 Asp Fr Arg Arg Gly Arg Cln Ile Tyr Ser Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu CAC CGC ASS CCC CGC CCC CAC ATC TAC TCC CCG TAC CAG ACC CTG GM CTG GAG MG GM 713 130 7 'A Met Arg Ser Ser Gly Thr Asp SrgLys Arg Gly Arg Gln Thr Tyr Thr Arg Tyr Gln Thr 80 90 ATG CGA ASC TCA GGA ACT GAC GC MA CGA GGCC CC CAG ACC TAC ACC CGC TAC CAG ACC 546 A Phe His Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile Ala Asn Ala Leu Cys 150 160 TTT CAC TTC MT CCC TAC CTA ACC CGC CGC CCC CCC ATC GAG ATC CCC MC GCG CTT TGC 773 Leu Clu Leu clu Lys clu Phe ills Tyr Asn Arg Tyr Leu Thr Arg Arg Ar8 Arg lle Siu CTG GAG CTG GAG MG GM TTT CAC TAC MT CGC TAC CTG ACC CCG CGC CGC CGC ATC SAG 606 100 110 Leu Thr Clu Arg Sln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Glu 170 180 CTG ACC GAG CGA CAC ATC MAA ATC TCG TTC CAG MC CGC CGC ATG MG TGG MA AMA GM 833 Ile Ala His Ala Leu Cys Leu Thr Sic Arg GIn Ile Lys Ile Trp Phe Gin Asn Arg Arg AIC GCCGCAC GCG CTC TGC CTC ACG GM AGA CAG ATC MC ATT TCC TTT CAG MC CCG CGC 666 120 130 Ser Asn|Leu Thr Ser Thr Leu Ser Gly Gly Gly Gly Sly Ala Thr Ala Asp Ser Met Gly TCT MT CTC ACA TCC ACT CTC TCG GGC GCC CCC GSA GCC CCC ACC CCC GAC AGC ATG CCC 893 A Met Lys Trp Lys Lys Glu Acn Thr Pro Gly Thr Thr Gly Cln Asp Arg Ala Ly6 Ala Gly 150 ATC MG TGG MA MG GAG MC MSG ACC GCG GCC CCG GGG ACC ACC GGC CM GAC AGG GCT 726 140 A A Gly Lys Glu Clu Lys Arg Glu Glu Thr GSi Glu Clu Lys Gln Lys Glu Tnc 210 GGA AM GAG GM MG CGG GM GAG ACA GAA GAG GAG MG CAG AA GAG TGACCAGGACTGTCC 956 Glu Ala Clu Gl Sblu Glu Glu Glu Trm GM GCA GAG GAG GSM GAS GAC TCAGGGATGGAGMAGGGCAGAGGMGAGACATGAGMAGGCAGACG 797 CTGCCACCCCTCTCTCCCTTTCTCCCTCGCTCCCCACCAACTCTCCCCTMTCACACACTCTGTATTTATCACTGGCAC 1035

876 MTTCATGTGTTTTGATTCCCTAAMCMAATTACGCACTCAAACCTGCACCTCMAGTCACCTCTGGACCCCCTCCCT 1114

955 AAAAACAAMAAAACCCTATTTMXTMAGMACGAGTTTAAAAACATTTTTTMGGACGGACAACGAGAAATTTTGSTT 1193

1034 TTTCMCACTCAAAAAATAGTACCTATAGGMAATCTGTCACTTTCGGTTTTTTTGTACMTATCAAAACGACATTATC 1272

TACCTCTTCTGTAGCTTTCTGGMTTTACCTCCCCTTTTCTATGTTCCTATTGTMGGTCTTTCTAAAATCTTGCAGTT 1113 CCCACTCCTTCGACGCCCCCCACCCCCGCCCCGCCTGCACACACCCGCCCCCTGCCCGCTCGGGCCTCTSCTCMACGCC 1351

TTSTMGCCCTCTTTMTGCtCTCTTTGTCGACTCTGGCTCTGGACTMCCCTGTGGTTCCCTGCCCTCCTGTGCCTCC 1192 TCACGGCCCATGGCACCCCGCAGCCCCGGAGCGCASCCCCCGCCCTTGGCCCCACACCMCCCCMGGGCCTCCCCGCAG 1430

GCCTTCCCAGCAGCGGCACCMAGCGCCTTAGGGAGCCCCAMMCCTACCACTCGCGTGTTCCCCMGCCCCTTCCTGC 1271 A CCCTGCCTAGCCCCTCTGCCCGACCMATGCCCASCCMGCAAATTCTATTTAAAGATCCTAGGGCTCATTATGGCAT 1509 TGCTGCTTGCTTCCCCTCCCCCAGCCCCATGCTCCCTTTACATTCTGTGTGTATCTAAAGCATGGAMMTAAAACCCA 1350 TTTACAAACTGTGACCGTTTCTGTGTGMGATTTTTAGCTGTATTTGTGGTCTCTGTATTTATATTTATGTTTAGCACC 1588 ATTAAAJMAT GTCAGTGTTCCTATCCMTTTCAAAAAACCMMAMACAGGGGAAATTACAAAAGAGAGMAAAAAAGTCMTCACGT 1667

B X HG 95.1 TTSTTTAGCCACTACCACAAAATAAATMAATAAAT X HG 125.3 B BgBgEEBgH3Bg H3 E H3 Bg A HG 8 A HG 65 1 kb STT S AT A EE E BE Bg Bg Bg BgEBgE EEBg E AAA 5 ---- _I ---- 3i 1 kb FIG. 1. (A) Nucleotide sequence of the human cDNA clone HHO.cl.95 and the conceptual translation of its longest open read- A Gy¶UA A 'AA ing frame. The region encoding the homeodomain is boxed. The second conserved homeodomain and the polyadenylylation signal (AATAAA) preceding the poly(A) tail are underlined. -*, Methionine 100 bp (ATG) codon representing the putative translation start site; *, putative glycosylation site; Trm, translation termination codon; y7, FIG. 2. (A) Nucleotide sequence of the human cDNA clone position ofthe intron; A, Hae III restriction sites used for subcloning HHO.c8.5111 and the translation of its longest open reading frame. HHO.cl.95. (B) Restriction map ofthe HHO.cl.95 transcription unit Symbols are as in Fig. LA. (B) Restriction map ofthe genomic region on two genomic clones (X95.1 and X125.3) as determined by Southern corresponding to HHO.c8.5111, as determined by Southern blot blot analysis. Open boxes indicate exons, and closed boxes indicate analysis on two phage clones (X8 and X65). Open and closed boxes homeobox sequences. E, EcoRI; Bg, Bgl II; A, Alu I; H3, HindIII; indicate exons and homeobox sequences, respectively. E, EcoRI; T, Taq I; S, Sma I. Bg, Bgl II; A, Alu I. 4916 Developmental Biology: Simeone et al. Proc. Natl. Acad Sci. USA 84 (1987) ysis is clearly necessary to assess the significance of this starting from the ATG codon at position 483 produces a peptide observation. of 153-amino acid residues containing a class I homeodomain HHO.cl.95 was used as a hybridization probe to screen a followed by 34 amino acids before the termination codon. The human genomic library in X Charon 28 by standard proce- 761-bp 3'-untranslated region contains a polyadenylylation dures. Two overlapping genomic clones, X95.1 and X125.3, signal 15 nucleotides upstream from the poly(A) tail, which is which contain a region of -24 kilobases (kb) corresponding followed by two additional and partially overlapping AATAAA to HHO.cl.95, were characterized by restriction endonucle- sequences. The original clone HHO.c8 is identical to ase mapping (Fig. iB) and were partially sequenced (not HHO.c8.5111 from position 594 to the poly(A) tail. shown). The HHO.cl.95 coding region is interrupted by an The c8 homeodomain is identical to that found in the cDNA intron of -2400 bp located upstream from the homeobox clone AC1 from Xenopus laevis (7). The homology between (Fig. 1 A and B). Two additional homeobox sequences appear the predicted peptide sequences derived from HHO.c8.5111 to be present in X95.1 upstream from the cl gene (Fig. 1B). and AC1 is further extended outside the homeodomain (22). Chromosome mapping by standard procedures (somatic cell In particular, a potential glycosylation site is present at the hybrids and in situ hybridization) indicates that the cl gene is 61st amino acid residue in both homeodomains (Fig. 2A; cf. localized in the long arm of chromosome 17 (K. Hubner, ref. 7). personal communication), possibly within the gene cluster Fig. 2B shows the genomic region corresponding to previously identified (13, 14) as containing Hul (termed c1O in HHO.c8.5111 obtained from two overlapping phage clones refs. 24), HO2, and HuS (18). Preliminary restriction data on (X8 and X65). Restriction analysis and partial sequencing (not cosmid clones locate the cl gene in a position corresponding to shown) indicate that the HHO.c8.5111 coding region is Hox2.3 within the Hox2 murine cluster (17). interrupted by an intron of -800 bp located upstream from The nucleotide sequence ofthe HHO.c8.5111 insert is shown the homeobox (Fig. 2A and B). An intron of 11 kb is present in Fig. 2A. Translation of the longest open reading frame further upstream in the 5'-untranslated region (Fig. 2 A and A Embryos Tissues (7wk)

*3 0.b o( *7t

c * 02 (0 A% I I IZ :A.

- 28 S

28S- ..~;t. -18S 18S- 1.6- qw p 1.6- io

MJ P--Actin

B FIG. 3. (A) (Left) Blot-hybridization Spinal cord Backbone Limbs Skin analysis of the expression of HHO.cl in poly(A)+ RNA from whole human embry- 6 7 8 7 8 6 7 8 7 8 9 os at 5, 6, and 7 weeks after fertilization. The relative mobilities of 28S and 18S rRNA are shown in size markers. Sizes are in kb. (Right) Expression of HHO.cl in -28 S poly(A)+ (2-3 ,ug per lane) and total skin -28S -28S -_28S (10 tmg) RNA obtained from pools oforgans or body parts dissected from human embryos at 7 weeks except for hearts, which were pooled from 7- to 8-week embryos, and skin, which was obtained at 9 weeks. -18 S -18 S -18S 16 Control, poly(A)+ RNA from an embryo 1.6- O 1.6- la 1.6- *W deprived of limbs and spinal column. (B) Expression of HHO.cl transcripts in poly(A)+ RNA (2 ,ug per lane) from spinal cord, backbone, and limbs and also in total -.mmXj RNA (10 ,ug per lane) from skin in human embryos from 6 to 9 weeks after fertiliza- __ tion, as analyzed by blot hybridization. Act in- A1ML.&fi-&.,dffl_ ___ Sizes are in kb. rRNA (28S and 18S) are shown as size markers. Developmental Biology: Simeone et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4917 B). The c8 gene has been mapped on the long arm of human RNA. As shown in Fig. 3A, the expression of cl transcripts chromosome 12 (K. Hubner, personal communication), in a was tissue-specific in 7-week embryos: 1.6-kb messages were position unrelated to previously identified homeobox gene abundant in spinal cord and detectable in backbone rudi- clusters on chromosomes 2 (ref. 25, and K. Hubner, personal ments and limbs. Virtually no hybridization signal was communication), 7 (35), and 17 (ref. 14 and this paper). observed in brain, heart, and liver, whereas a barely detect- The putative proteins of HHO.cl.95 and HHO.c8.5111 able band was still present in a control embryo deprived of contain a conserved pentapeptide Ile-Tyr-Pro-Trp-Met that limbs and spinal column. A low signal at 1.6 kb was also starts at amino acid position -11 and -19, respectively, from detectable in total RNA from skin, together with a broad band the homeodomain (25). of cross-hybridization to 28S rRNA. We have utilized HHO.cl and HHO.c8 as cDNA probes to The expression of cl in positive tissues was then analyzed analyze the expression of the cl and c8 genes in human at 6 to 9 weeks by blot-hybridization analysis of poly(A)+ embryonic development. (spinal cord, backbone, and limbs) or total (skin) RNA (Fig. Blot-hybridization analysis of poly(A)+ RNA obtained 3B). The abundance of 1.6-kb transcripts shows a 3- to 5-fold from whole embryos at 5, 6, and 7 weeks indicates that cl is increase in spinal cord and backbone rudiments at 6 to 8 and expressed as a single major transcript of 1.6 kb at a roughly 7 to 8 weeks, respectively; a nearly constant low level was constant level throughout this period (Fig. 3A). No signal was observed in limbs and skin at 6 to 8 and 7 to 9 weeks. detected by blot hybridization ofthe corresponding poly(A)- The expression of c8 gene was analyzed on the specimens RNAs (not shown). described above. HHO.c8 detected two transcripts of2.2 and We have then analyzed the expression of cl in different 1.8 kb by blot hybridization of poly(A)+ RNA from whole organs and body parts of 7-week embryos. Samples from embryos at 5, 6, and 7 weeks at a roughly constant level (Fig. different specimens were pooled when necessary. Hearts 4A Left). No signal was detected by blot hybridization of from 7- and 8-week embryos were pooled. The central corresponding poly(A)- RNAs (not shown). The 2.2- and nervous system was obtained free of contaminating tissues; 1.8-kb transcripts are abundantly accumulated in spinal cord brain was dissected from spinal cord and medulla oblongata with a 1:2 ratio. The 1.8-kb RNA is predominantly expressed at the level of the pontine flexure. Skin was analyzed as total at a high level in limbs and backbone rudiments and at a lower A Embryos Tissues (7wk)

A 4* 04.. e

'b~~~~c~~~~ ~~ 4;, *4 cip *zt CO*t ell,~

28S- *- 28S- _ -28 S

--22 2.2- 18S- . 1.8 18S - -1.8 1 ota 1.8- a1 -18S

FIG. 4. (A) (Left) Blot-hybridiza- Wfo t-Actin tion analysis of the expression of HHO.c8 in poly(A)+ RNA (2-3 ,ug per lane) from whole human embryos at B 5, 6, and 7 weeks after fertilization. Spinal cord Backbone Limbs SkinSkin Sizes are in kb. rRNA (28S and 18S) are shown as size markers. (Right) 6 7 8 9 7 8 6 7 8 7 B 9 Expression of HHO.c8 in poly(A)+ (2-3 ,g) or total skin (10 ,ug) RNA from pools of organs or body parts dissected from human embryos at 7 28S- -28 S _28S weeks except for hearts, which were pooled from 7- to 8-week embryos, and skin, which was obtained at 8 weeks. Control, poly(A)+ RNA from 22_ an embryo deprived of head, limbs, - 18S 1 2.2 - %, -18S 8- _ 18S and column. 1.8 -*. s 22- heart, liver, spinal (B) 1.8- Expression of HHO.c8 as analyzed by blot hybridization of poly(A)+ RNA (2 ,ug per lane) from spinal cord, backbone rudiments, and limbs and also from total RNA (10 ,g per lane) from skin obtained from human embryos from 6 to 9 weeks after concep- Actin- *Se Sizes are in kb. rRNA and __w as me___ tion. (28S 18S) are shown as size markers. 4918 Developmental Biology: Simeone et al. Proc. Natl. Acad. Sci. USA 84 (1987) level in heart (Fig. 4A Right). No signal was observed in brain more specific functions may be exerted by the nonconserved and liver. Both bands are clearly detected by blot hybridiza- parts of homeobox gene proteins, which diverged in various tion of skin total RNA (together with a prominent band of evolutionary lineages. cross-hybridization to 28S rRNA) at levels comparable to We thank Dr. W. J. Gehring for providing the Drosophila probes, those observed in spinal cord or limbs. Dr. H. K. Okayama for the cDNA library, and Dr. G. Cossu for A 1.8-kb band was still detected in poly(A)+ RNA from a helpful discussions. The expert assistance of Mrs. P. Alessandri in control embryo upon removal of head, limbs, spinal column, typing the manuscript is gratefully acknowledged. This work was liver, and heart. Although at least part of this signal may be supported by grants from Consiglio Nazionale delle Richerche, accounted for by the presence of skin, it cannot be ruled out Rome, Progetti Finalizzati "Ingegneria Genetica e Basi Molecolari that c8 transcripts are expressed in other organs or tissues. delle Malattie Ereditarie" to E.B. and C.P. (Grant No. 85.01425.51), Analysis of c8 expression at different stages of develop- "Oncologia" to E.B., F.M. (85.02244.44), and C.P. (85.02560.44), ment indicated that the abundance of both 1.8- and 2.2-kb "Medicina Preventiva" to C.P. (85.00707.56), and the Italian Asso- transcripts, as well as their relative ratio, is nearly constant ciation for Cancer Research (AIRC) to E.B. from 6 to 9 weeks in spinal cord, 6 to 8 weeks in limbs, 7 to 1. Gehring, W. (1985) Cold Spring Harbor Symp. Quant. Biol. 50, 243-251. 8 weeks in backbone rudiments, and 7 to 9 weeks in skin (Fig. 2. Lewis, E. B. (1978) Nature (London) 276, 565-570. 3. Kaufman, T. C., Lewis, R. & Wakimoto, B. (1980) Genetics 94, 115-133. 4B). 4. McGinnis, W., Levine, M. L., Hafen, E., Euroiwa, A. & Gehring, W. J. In order to exclude artifacts due to cross-hybridization to (1984) Nature (London) 308, 428-433. transcripts derived from other homeobox genes, we have 5. Scott, M. P. & Weiner, A. J. (1984) Proc. Natl. Acad. Sci. USA 78, hybridized blots of spinal cord poly(A)+ RNA to different 1095-1099. 6. Dolecki, G. J., Wannakrairoj, S., Lurn, R., Wang, G., Riley, H. D., subclones of both inserts. In particular, we utilized two Carlos, R., Wang, A. & Humphreys, T. (1986) EMBO J. 5, 925-930. noncontiguous Hae III fragments containing the homeobox 7. Carrasco, A. E., McGinnis, W., Gehring, W. J. & De Robertis, E. M. and the 3'-translated and untranslated regions of HHO.cl.95, (1984) Cell 37, 409-414. as well as two contiguous Hae III fragments containing the 8. Mueller, M. M., Carrasco, A. E. & De Robertis, E. M. (1984) Cell 39, 157-162. homeobox and the 3'-translated and untranslated regions of 9. Harvey, R. P., Tabin, C. J. & Melton, D. A. (1986) EMBO J. 5, HHO.c8.5111 (see Figs. lA and 2A). In both cases, hybrid- 1237-1244. ization to all subclones detected exactly the same bands (not 10. McGinnis, W., Hart, C. P., Gehring, W. J. & Ruddle, F. H. (1984) Cell shown). Furthermore, these data indicate that both the 2.2- 38, 675-680. 11. Colberg-Poley, A. M., Voss, S. D., Chowdhury, K. & Gruss, P. (1985) and 1.8-kb transcripts detected by HHO.c8 have essentially Nature (London) 314, 713-718. the same structure in the region spanning the homeobox and 12. Colberg-Poley, A. M., Voss, S. D., Chowdhury, K., Stewart, C. L., the proximal 3'-untranslated sequences, thus suggesting that Wagner, E. F. & Gruss, P. (1985) Cell 43, 39-45. they might differ in their 5' portions. 13. Rabin, M., Hart, C. P., Ferguson-Smith, A., McGinnis, W., Levine, M. & Ruddle, F. H. (1985) Nature (London) 314, 175-178. We conclude that cl and c8 are differentially expressed and 14. Joyner, A. L., Lebo, R. V., Kan, Y. W., Tjian, R., Box, D. R. & developmentally regulated in human embryogenesis. More- Martin, G. R. (1985) Nature (London) 314, 173-175. over, their expression patterns differ from those of other 15. Joyner, A. L., Kornberg, I., Coleman, K. G., Cox, D. R. & Martin, human (24, 25) and mouse (12, 15-18, 20) homeobox genes G. R. (1985) Cell 43, 29-37. 16. Jackson, I. J., Schofield, P. & Hogan, B. (1985) Nature (London) 317, reported so far. 745-748. The pattern of expression observed for cl and c8, as well 17. Hart, C. P., Awgulewitsch, A., Fainsod, A., McGinnis, W. & Ruddle, as cdO and c13 (24, 25), allows the formulation of a series of F. H. (1985) Cell 43, 9-18. general concepts: (i) The four genes are differentially ex- 18. Hauser, C. A., Joyner, A. L., Klein, R. D., Learned, T. K., Martin, G. R. & Tjian, R. (1985) Cell 43, 19-28. pressed according to tissue- and/or stage-specific patterns. 19. Awgulewitsch, A., Utset, M. F., Hart, C. P., McGinnis, W. & Ruddle, Most of them encode multiple transcripts. (ii) All are abun- F. H. (1986) Nature (London) 320, 328-335. dantly transcribed in embryonic spinal cord, which repre- 20. Duboule, D., Baron, A., Mihl, P. & Galliot, B. (1986) EMBO J. 5, sents a typical metameric structure. In particular, strong 1973-1980. 21. Levine, M., Rubin, G. N. & Tjian, R. (1984) Cell 38, 667-673. heterogeneous 2.1- and 2.5-kb bands are detected in spinal 22. Boncinelli, E., Simeone, A., La Volpe, A., Faiella, A., Fidanza, V., cord by HHO.clO (24) and HHO.c13 (25), respectively. Acampora, D. & Scotto, L. (1985) Cold Spring Harbor Symp. Quant. Clone HHO.cl detects 1.6-kb transcripts of the same size Biol. 50, 301-306. class, which are also present in other embryonic tissues in 23. McGinnis, W. (1985) Cold Spring Harbor Symp. Quant. Biol. 50, 263-270. lower abundance. Clone HHO.c8 detects two bands of 2.2 24. Simeone, A., Mavilio, F., Bottero, L., Giampaolo, A., Russo, G., and 1.8 kb with a specific intensity ratio different from that Faiella, A., Boncinelli, E. & Peschle, C. (1986) Nature (London) 320, observed in other embryonic tissues. (iii) All genes, with the 763-765. possible exception of cdO, are transcribed in backbone 25. Mavilio, F., Simeone, A., Giampaolo, A., Faiella, A., Zappavigna, V., Acampora, D., Poiana, G., Russo, G., Peschle, C. & Boncinelli, E. rudiments. (iv) cl, c8, and cdO do not detect any transcripts (1986) Nature (London) 324, 664-668. in embryonic brain throughout the analyzed period. c13 is 26. Migliaccio, G., Migliaccio, A. R., Petti, S., Mavilio, F., Russo, G., expressed as a single 4.2-kb band in mesencephalon and Lazzaro, D., Testa, U., Marinucci, M. & Peschle, C. (1986) J. Clin. metencephalon (not in prosencephalon) at 6 to 7 weeks but Invest. 78, 51-60. not at 27. Moore, K. L. (1982) The Developing Human (Saunders, Philadelphia). 8 weeks or later. (v) Although the expression of cl and 28. Peschle, C., Mavilio, F., Care, A., Migliaccio, G., Migliaccio, A. R., cdO is, respectively, largely and exclusively restricted to Salvo, G., Samoggia, P., Petti, S., Guerriero, R., Marinucci, M., spinal cord (and apparently backbone), c8 and c13 are also Lazzaro, D., Russo, G. & Mastroberardino, G. (1985) Nature (London) expressed in a large spectrum of embryonic structures (limb 313, 235-238. buds or 29. Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289. limbs, heart, skin, etc.), which follow dramatically 30. Garber, R. L., Kuroiwa, A. & Gehring, W. J. (1983) EMBO J. 2, different patterns of morphogenesis. (vi) Homeobox tran- 2027-2036. scripts have not been detected so far in a variety of organs or 31. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A body parts (e.g., liver). Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring In genes Harbor, NY). conclusion, the concept emerges that in humans 32. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, containing homeodomains may play a wide spectrum of 560-564. control activities in early development of different organs or 33. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412. to a 34. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. body parts. According unified model, the homeodomain (1979) Biochemistry 18, 5294-5300. might exert a general function (possibly DNA-binding), 35. Bucan, M., Yang-Feng, T., Colberg-Poley, A. M., Wolgemuth, D. K., which is necessarily shared by all homeobox gene products; Guenet, I. L., Francke, V. & Lehrach, H. (1986) EMBO J. 5, 2899-2905.