Proc. Natl. Acad. Sci. USA

Vol. 88, pp. 2427-2431, March 1991 Developmental Biology

Quox-1, a quail homeobox gene expressed in the embryonic central nervous system, including the forebrain (homeodomain/development/cDNA/genoinic DNA/in situ hybridization)

ZHI-GANG XUE*t, WALTER J. GEHRINGf, AND NICOLE M. LE DoUARIN* *Institut d'Embryologie Cellulaire et Moldculaire du Centre National de la Recherche Scientifique et du College de France, 49 bis, Avenue de la Belle

Gabrielle, 94736 Nogent-sur-Mame Cedex, France; and tDepartment of Cell Biology, Biozentrum, University of Basel, Kingelbergstrasse 70, CH-4056 Basel, Switzerland

Contributed by Nicole M. Le Douarin, December 3, 1990

ABSTRACT

This paper reports the cloning and se-

quencing of a quail homeobox-containing gene, Quox-1, and its expression pattern in embryos from 3 to 6 days (E3 to E6) of development as determined by in situ hybridization. The opening reading frame in cDNA clone gil corresponds to a predicted protein of .242 amino acids. Quox-1 protein displays high sequence similarity to the Antennapedia family, especially to the mouse homeodomain-containing protein Hox-l.1 (100% identity in the homeobox region, 77% at the 5' end beyond the homeobox). However, the carboxyl-terminal domain of the postulated protein has no significant homology with other known homeoproteins, including Hox-l.l. In situ hybridization experiments showed that Quox-1 is widely expressed in the developing central nervous system including the entire brain and the spinal cord. Outside the central nervous system, transcription of Quox-1 was mainly detected in the endodermderived epithelium of esophagus, trachea, and other digestive organs, as well as in the sensory epithelium of the olfactory region and perichondrium of the vertebrae. Thus, Quox-1 transcripts have a remarkably wide distribution that, unlike the other vertebrate homeobox genes examined to date, encompasses the rostral part of the developing nervous system, including the forebrain. Genes characterized by a highly conserved sequence of 180 nucleotides called the homeobox are considered to play an important role in controlling the establishment of the body

plan during the development of Drosophila (1-3). The 60 amino acids encoded by the homeobox constitute a homeodomain conferring on the proteins in which it is present the capacity to bind specific DNA sequences, and therefore the potential ability to regulate the expression of certain genes during embryogenesis (4-7). Following this discovery, much research has been initiated with the goal of finding similar genetic elements in vertebrates. So far homeobox-containing genes have been identified in amphibians (8), fish (9), birds (10), mammals (2), and humans (11). The mouse Hox genes have attracted much attention and their spatiotemporal expression patterns have been analyzed by in situ hybridization. As in Drosophila, their transcription seems to be restricted to a precise window during development and in a limited set of differentiating cells, thus suggesting a role in the patterning of development. Ectopic expression of Hox-J.J (12), overexpression of Hox-1.4 in transgenic mice (13), and overexpression of Xhox3 by injection of synthetic RNA into Xenopus embryos (14) have been found to result in segmental transformations and other developmental abnormalities. Interference with the function of another homeobox gene (Xl14box 1) in Xenopus embryos by injecting the corresponding antibodies leads to the transfor-

mation of the anterior spinal cord into a hindbrain-like structure (15). However, largely because of the difficulties encountered in performing genetic and experimental analysis of embryogenesis in vertebrates, the precise functional role of these gene products remains elusive. Mammals and birds have very similar developmental strategies, but as an experimental model, the avian embryo presents a number of practical advantages. Embryos can be manipulated from very early stages of development onward and, at the phenomenological level, cell-cell interactions can be analyzed with great refinement during the establishment of body plan and organogenesis in this class of vertebrates. Combining information yielded by the highly patterned expression of developmental genes with that provided by embryonic manipulations may constitute a favorable approach to understanding how genes control development. This is one of the reasons for which attempts are now being made to identify homeobox-containing genes in birds. Some have already been detected in chicken or quail (10, 16-18) on the basis of their homology with mouse or Drosophila genes. The second reason for such an undertaking is phylogenetic; comparative analysis of corresponding genes in different representatives of the animal kingdom can provide valuable information on their importance during development. With this in mind we have isolated and sequenced a cDNA and a gene from the quail that we have called Quox-1 because of its strong homology with the Hox-J .1 gene of the mouse. We report here its expression pattern in 3- to 6-day (E3-E6) quail embryos.§

MATERIALS AND METHODS cDNA Cloning. A cDNA library was constructed in Agtl0 (19) with RNA prepared from E5 quail spinal cords by the LiCl/urea extraction method (20). This cDNA library was screened using 32P-labeled p903G (BamHI-Pvu II fragment) including the homeobox of the Antennapedia (Antp) gene of Drosophila under conditions of reduced stringency as described (2). Four positive clones were isolated after screening a nonamplified library of 3 x 105 cDNAs. One clone, c3, containing a 360-base-pair (bp) insert, was named Quox-1. The other three clones isolated independently contained an identical sequence. To isolate a longer Quox-1 cDNA clone (gil), a second cDNA library from E6 quail was constructed in a AZAP phagemid vector (Stratagene) according to Haymerle et al. (21). An oligonucleotide probe corresponding to the 5' region of the Quox-1 homeobox (CGCCAGACCTACACCCGCTACCAGACCCTGGAG) was used to screen the cDNA library under conditions described by Wood et al. (22). Abbreviations: CNS, central nervous system; En, embryonic day n. tTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. M59714).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2427

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Proc. Natl. Acad. Sci. USA 88 (1991)

Developmental Biology: Xue et al.

Isolation and Mapping of Genomic Clones. A library was constructed by partial digestion with Mbo I of genomic DNA prepared from blood, followed by cloning into EMBL3 bacteriophage. The cDNA fragment (clone c3) was used to screen 5 x 105 recombinants of the genomic library under high-stringency conditions [50% formamide/4x SSC (ix is 0.15 M NaCI/0.15 M sodium citrate, pH 7.0)/0.02% Ficoll/ 0.02% polyvinylpyrrolidone/0.02% bovine serum albumin/ 0.1% SDS with denatured salmon sperm DNA at 100 jug/ml; 420C for 12 hr]. The genomic clones were mapped by single and double digestion with restriction enzymes. Hybridization to Quail Genomic DNA. For Southern analysis, 20 ,ug of genomic DNA was cleaved with restriction enzymes, separated in a 0.8% agarose gel, and blotted by standard procedures. Hybridization was carried out under high-stringency conditions as described above, with washes at 650C in 0.lx SSC/0.1% SDS. Northern Blot Analysis. Thirty micrograms of total RNA from each stage was electrophoresed in 1% agarose/2.2 M formaldehyde gel and subsequently transferred to nitrocellulose membrane in 20x SSC. Hybridization and wash stringencies were the same as for Southern blot analysis. DNA Sequencing. Positive clones were subcloned into the plasmid pBluescript (Stratagene) and sequenced as single- or double-stranded DNA by the dideoxy nucleotide method (Sequenase Kit, United States Biochemical). Each sequence was determined at least twice on independent cDNA and genomic clones. Preparation of Embryo Sections. Quail embryos from E3-E6 were fixed in 2% paraformaldehyde in phosphatebuffered saline for 1-12 hr. Then they were placed in phosphate-buffered saline with 15% sucrose overnight, dehydrated, and put into paraffin. Sections of 5 to 7 pum were mounted on gelatin-coated slides. In Situ Hybridization. This was carried out on paraffin sections as described (23). The RNA probe was produced by using T3 or T7 polymerase (Promega) to transcribe DNA templates derived by insertion of the cDNA fragment into pBluescript. Uridine 5'-[a-[35S]thio]triphosphate (1000 Ci/ mmol, Amersham; 1 Ci = 37 GBq) was used as substrate.

Sequence comparison with other homeobox genes reveals that Quox-1 clearly belongs to the Antp class (83% and 98% sequence identity with the Antp homeobox for the DNA and protein sequences, respectively). The homeobox sequence shows 87% identity to that of mouse Hox- .1 (24) and 82% to Xenopus Xhox36 (26) at the nucleotide level (data not shown) and 100% identity to both at the protein level (Fig. 3). This homology extends outside the homeobox at the 5' end; 77% of the amino acids and 76% of the nucleotides of Quox-1 (data not shown) are identical with Hox-1.1 (Fig. 3). In contrast, the 3' domain outside the homeobox does not have any significant homology with other known homeodomain proteins, including Hox-1.1. Another feature of Quox-1 cDNA is of interest: in the 3' region starting at position 737 (underlined in Fig. 1), one 12-bp sequence (AAGTGGAAGAAG) repeats perfectly a highly conserved sequence of the DNA-recognition region in the homeodomain (positions 55-58; refs. 27 and 28). Expression of Quox-1 During Early Embryonic Development. Two cDNA fragments (al and b2) that correspond to the first and second exons of the Quox-1 genomic DNA (Fig. 2) were used as probes. Hybridization of cDNA al or b2 with cleaved genomic DNAs under stringent conditions revealed hybridization to fragments of the sizes expected from the

RESULTS Quox-l Complementary and Genomic DNA. The nucleotide sequence and deduced protein sequence of Quox-1 (gil) are presented in Fig. 1. The comparison with the genomic sequence revealed an intron of about 1.1 kilobases (kb) located 2 codons upstream of the homeobox (Figs. 1 and 2). The perfect splice consensus sequence (CAGIGT) was found in donor and acceptor sequences. The homeobox is located in the second exon (Fig. 2). This structure is quite similar to the genomic DNA of murine Hox-J.1 (24). Quox-1 cDNA (gil) contains an open reading frame of 242 amino acids (33,671 daltons) with the predicted homeodomain in the 3' half of the cDNA. The first ATG codon after two in-frame stop codons is the most probable initiator site since its context fulfills Kozak's rule (25). As in most homeobox genes, a conserved hexapeptide sequence, IleTyr-Pro-Trp-Met-Arg, is encoded 5 amino acids upstream of the homeobox. A stop codon at position 836 terminates the open reading frame. There is a potential polyadenylylation signal (ATTAAAA) at position 887 in the 3' untranslated region. Clone gil has no proper poly(A) tail; comparison with the genomic sequence showed that the 15 continuous adenosine residues are present in the genomic DNA. Thus, it is conceivable that the gil sequence was initiated at an internal A-rich region able to pair with an oligo(dT) primer rather than at the terminus of Quox-1 mRNA. Unlike many other homeodomain proteins, which are rich in proline, the predicted Quox-1 protein is rich in serine (14%) and arginine (10%).

1

GTCCAAAAGAAAATGGGGTTTGGTGTAAATCTGGGGGAGTAATGTTATCATATATCAC

59

GCTACCTCGTAAACCGACACTGAAGCTGCGACAACAATCACGGTCAAAAT

116

TCT TCG TAT TAT GTG AAT GCG CTT TTT AGC AAA TAT ACG GCG GGG Ser Ser Tyr Tyr Val AMn Ala Lou Phe Ser Lys Tyr Thr Ala Gly

161

GCT TCT CTT TTC CAA AAT GCA GAG CCT ACT TCT TGC TCC TTT GCA Ala Ser Lou Phe Gln AMn Ala Glu Pro Thr Ser Cys Ser Phe Ala

206

AGC AAC TCC CAG AGA AGC GGC TAT GGA CCA GGC GCG GGT GCC TTC Sor AMn Ser Gln Arg Ser Gly Tyr Gly Pro Gly Ala Gly Ala Phe

251

GCC TCC TCC ATG CCC GGC TTG TAC AAT GTG AAC AGT ACC ATA TAT Ala Ser Ser Not Pro Gly Leu Tyr Asn Val Mn Ser Thr Ile Tyr

296

CAA AGC CCG TTC TCT TCC GGA TAC AGC CTG GGC TCT GAT GCC TAC Gln Sr Pro Phe Ser Ser Gly Tyr Ser Lou Gly Ser Asp Ala Tyr

341

AAC CTG CAT TGT TCC TCT TTT GAT CAG AAC ATT CCC GTC CTC TGC AMn Lou His Cys Scr Ser Phe Asp Gln AMn Ile Pro Val Lou Cys

386

AAT GAC CTA ACC AAA CCC AGC TGT GAG AAA GCG GAG GAA AGG CAA AMn Asp Lou Thr Lys Pro Ser Cys Glu Lys Ala Glu Glu Arg Gln

431

CCT GCA CAA CCA GGT CGA GGT CAA ?TT CGG TAC CCC TGG ATG Pro Ala Gln Pro Gly Arg Gly Gln Phe Arg Ile Tyr Pro Trp Not

476

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CGC TAC CAG ACC CTG GAG CTG GAG AAG GAA TTC CAC TTC AAT CGC

Arg Tyr Gln Thr Lou Glu Lou Glu Lys Glu Phe His Phe Asn Arg 566

TAT CTG ACT CGG CGG CGG CGG ATA GAG ATC GCC CAT GCC CTC TGC

Tyr Lou Thr Arg Arg Arg Arg Ile Glu Ile Ala His Ala Lou Cys 611

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656

MAMC ,Lys Tra

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hn MQ ATG AAG AAG AGG AGG ATT AGG GAG TCC CAT TCG AGG AAG CAA Ly Met Lys Lys Arg Arg Ile Arg Glu Scr His Sr Arq Lys Gln TCT CT? GAG ATA ACG TAT GCA CGT GAC AGC TGT AAA AGC TCC CTT Scr Lou Glu Ile Thr Tyr Ala Arg Asp Sox Cys Lys Ser Ser Lou AGTTATTAAAAG AAAAAAAAAAAAAAA

FIG. 1. Primary structure of Quox-l cDNA clone gil and encoded protein. The sequence of the putative Quox-l protein is shown below the open reading frame. Identical sequences were obtained over the relevant regions with the c3 cDNA clones, gil cDNA clone, and the genomic clone. The splice site is indicated by a solid triangle, and the boxes outline the conserved hexapeptide and the homeodomain. The repeated sequences are underlined.

Proc. Natl. Acad. Sci. USA 88 (1991)

Developmental Biology: Xue et al. 5 E

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FIG. 2. Schematic representation of the shared intro-exon arrangement and the location of the Quox-1 homeobox (filled box) on genomic DNA and cDNA. The homeobox location (filled box), the probes used for Northern blot, Southern blot, and in situ hybridization, and the oligonucleotide probe used to isolate the cDNA are shown. B, Bgl II; E, EcoRI; Pv, Pvu II; S, Sac I.

genomic clone in each case (Fig. 4b), showing that the sequences in Quox-1 DNA detect a single locus. RNA extracted from E4-E6 embryos was analyzed by Northern blotting. In view of the extensive sequence identity between Quox-1 and other homeobox genes, a cDNA fragment (b2) that contains the specific carboxyl-terminal region and 3' untranslated region was used as a probe (Fig. 2). Only one major transcript (2.0 kb) was detected in RNA from E4-E6 embryos (Fig. 4a). The length of the gil cDNA is about 1 kb, thus confirming that gil is only part of the full-length transcript. We used in situ hybridization to investigate the spatial pattern of Quox-1 gene expression during quail embryo development. Two probes derived from Quox-1 were used, al (a fragment 5' of the homeobox) and b2 (used for the Northern blot) (Fig. 2). These two RNA probes gave the same expression pattern in in situ hybridization experiments. As negative control we used a sense-strand probe complementary to the al and b2 sequences. In no case with this control and under the conditions used did we detect spurious hybridization. Whole embryos from E3-E6 were examined. In most cases, embryos were cut in parasagittal sections. We chose these stages because they correspond approximately to mouse embryos 11-16 days postconception (29), in which maximal expression of most murine homeobox genes is observed. By E3, Quox-1 transcripts were detected mainly in the central nervous system (CNS). Most of the transcripts were localized in the neural tube and the entire brain, including the telencephalon, hypothalamus, and the optic stalk (Fig. 5a).

In the E5-E6 quail, the dorsal position of the hemispheres have grown considerably and the walls of the brain have continued to thicken. The hybridization seen in the CNS was still at a high level. Notably, transcripts accumulated in the thick mantle layer of the brain. However, the density of grains was higher in the ventricular epithelium and subventricular zone than in the mantle layer (Fig. 5 b-d). With the cessation of neuroblast proliferation, a decrease of Quox-1 expression was observed in the spinal cord. Outside the CNS, expression of Quox 1 was detected in the neural crest-derived spinal ganglia (Fig. 5a), in the placodal ectoderm-derived sensory epithelium of the olfactory region, and in the endoderm-derived esophagus, trachea, liver, and epithelial cell layer of intestine and stomach. Transcripts were also visible in the perichondrium of vertebrae (Fig. 5 c and d).

DISCUSSION The Quox-1 gene, as analyzed by in situ hybridization, is widely expressed in the developing CNS, including all regions of the brain and spinal cord. This is a very special feature of this homeobox gene, since most Hox genes described in the mouse have a well-defined limit of expression, generally located anteriorily in the hindbrain (30, 31), except for mouse engrailed-related genes En-i and En-2, which are expressed at the junction of the developing midbrain with the hindbrain (32, 33). In contrast, Quox-1 is expressed in a much b

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221 SHSRKQSLEI-EEEEEEEEStop

FIG. 3. Comparison of the amino acid sequence of quail Quox-1 with those of mouse Hox-1.1, Xenopus Xhox36, and Drosophila Antennapedia. (Antp.) The homeodomain sequences are boxed and only the amino acids that differ from those of Quox-1 are shown below the predicted Quox-1 protein sequence.

1

2

1

2

3

4

5

6

FIG. 4. Northern and genomic Southern blot analysis with the al and b2 probes under stringent hybridization conditions. Sizes are shown in kilobases. (a) Northern blot of total RNA from E4 (lane 1) and E6 (lane 2) quail. Thirty micrograms of RNA was loaded in each lane. The blot was hybridized with the b2 probe and autoradiographed for 72 hr. Positions of 28S and 18S rRNA are indicated. (b)

Quox-1 hybridization to unique genomic fragments. Quail genomic DNA (20 ,ug) was digested with EcoRI (lanes 1 and 4), BamHI (lanes 2 and 5), or Sac I (lanes 3 and 6). Lanes 1-3 were hybridized with probe al; lanes 4-6 were hybridized with probe b2.

2430

Developmental Biology: Xue et al.

more rostral region of the developing CNS including eye, telencephalon, diencephalon, mesencephalon, metencephalon, myelencephalon, and spinal cord. To our knowledge, Quox-1 is the first vertebrate homeobox gene to be described in which expression occurs throughout the developing CNS. The transcription pattern of Quox-1 also shows temporal and spatial changes during neuroembryogenesis. As growth ofthe brain proceeds, Quox-1 mRNAs become increasingly abundant. At the same time, the spinal cord becomes more weakly labeled as neuronal proliferation decreases. This expression pattern suggests that, at these early stages, Quox-1 may be implicated in regulating cell divisions in the neural epithelium. Similar expression patterns have been reported for the POU-domain gene family in the mouse (34). For example, between days 13 and 16 of fetal development (corresponding to E4.5-E6 of the quail), the Brn-i and Brn-3 transcripts are widespread in the mouse CNS, including the telencephalon and diencephalon (34).

Proc. Natl. Acad. Sci. USA 88 (1991)

There are similarities in the expression of homeobox genes between vertebrates and Drosophila at comparable stages of development. Genetic studies have-shown that, in addition to their classical role in pattern formation, the products of the ftz (35), cut (36), and rough (37) genes in Drosophila are necessary for specification of neuronal identity in the development of the CNS. Thus, homeobox genes in vertebrates, as well as in invertebrates, may possibly act in controlling neuronal fate. The expression pattern of Quox-1 in the CNS makes it a good candidate for such a role. A high level of homology exists in the cDNA sequence and genomic DNA structure between the quail Quox-1 gene and the mouse Hox-1.I genes (24), but the quail gene transcripts are present more anteriorly than those of Hox-1.1 in the CNS (38). Whether conservation of structure reflects homologous function for homeobox genes in the development of different species has yet to be shown. In certain cases, similarities of structure go together with identical expression patterns in

S.

d FIG. 5. Localization of Quox-1 transcripts within parasagittal sections of E3 (a), E5 (b), and E6 (c and d) quail as shown by in situ hybridization with the b2 probe. Brightfield illumination was used for d and darkfield for a-c. T, telencephalon; D, diencephalon; M, mesencephalon; R, rhombencephalon; S, spinal cord; SG, spinal ganglion; NR, neuroepithelial layer of retina; 0, sensory epithelium of olfactory region; E, esophagus; Tr, trachea; ET, esophagus-trachea region; L, liver; P, perichondrium of the vertebrae.

Developmental Biology: Xue et al. mammals and birds, as in the case of Hox-7.1 and Quox-7 (18). Thus homology of function is a reasonable assumption as long as the function of the homeodomain of the encoded protein resides in its DNA-binding capacities. The specific role of homeotic proteins is likely to be specified also by other domains. In such a perspective, expression of Quox-1 in the CNS may be related to its specific 3' coding region, for which no significant homology with any known vertebrate homeobox genes was found except for a highly conserved fragment (Lys-Trp-Lys-Lys) that is also found in helix IV of the homeodomain (28). We are grateful to X. Jin-Xue for excellent technical assistance. We thank Dr. M. Buckingham and Dr. J. Smith for critical reading of the manuscript. We acknowledge the help of B. Henri, Y. Rantier, and S. Gournet in preparation of figures. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Mddicale, the Association pour la Recherche Contre le Cancer, and the Fondation pour la Recherche Mddicale Franraise. Z.-G.X. received a shortterm fellowship from the European Molecular Biology Organization. 1. McGinnis, W., Levine, M., Hafen, E., Kuroiwa, A. & Gehring, W. J. (1984) Nature (London) 308, 428-433. 2. McGinnis, W., Gaber, R. L., Wirz, J., Kuroiwa, A. & Gehring, W. J. (1984) Cell 37, 403-408. 3. Scott, M. P. & Weiner, A. J. (1984) Proc. Nati. Acad. Sci. USA 81, 4115-4119. 4. Shepherd, J. C. W., McGinnis, W., Carrasco, A. E., De Robertis, E. M. & Gehring, W. J. (1984) Nature (London) 310, 70-71. 5. Laughon, A. & Scott, M. P. (1984) Nature (London) 310, 25-30. 6. Hiromi, Y. & Gehring, W. J. (1987) Cell 50, 963-974. 7. Jaynes, J. B. & O'Farrell, P. H. (1988) Nature (London) 336, 744-749. 8. Carrasco, A. E., McGinnis, W., Gehring, W. J. & De Robertis, E. M. (1984) Cell 37, 409-414. 9. Eiken, H. G., Njolstad, P. R., Molven, A. & Fjose, A. (1987) Biochem. Biophys. Res. Commun. 149, 1165-1171. 10. Rangini, Z., Frumkin, A., Shani, G., Guttmann, M., EyalGiladi, H., Gruenbaum, Y. & Fainsod, A. (1989) Gene 76, 61-74. 11. Levine, M., Rubin, G. M. & Tjian, R. (1984) Cell 38, 667-673. 12. Balling, R., Mutter, G., Gruss, P. & Kessel, M. (1989) Cell 58, 337-347.

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