The Journal of , February 1994, 14(2): 466496

Asymmetric Expression of a Novel in Vertebrate Sensory Organs

David L. Deitcher,” Donna M. Fekete,b and Constance L. Cepko Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115

A novel homeobox gene, SOHo-1, was isolated from em- (Cohen and Jiirgens, 1990; Finkelstein and Perrimon, 1990). A bryonic chicken retina. On embryonic day 2 (E2), SOHo- 7 is growing list of homeobox genesare also known to be expressed expressed in the retina, posterolateral otic pit, and neural in the developing vertebrate brain (Lazzoro et al., 1991; Porteus tube anterior to the spinal cord. On E4, SOHo- 7 is expressed et al., 1991; Price et al., 1991, 1992; Simeoneet al., 1992a,b). at high levels in anterior retina and low levels in posterior Evidence is accumulating to support the notion that some retina, suggestive of a role in patterning the anterior-pos- homeobox genesin vertebrates act in analogousmanners to terior axis. It is also expressed on E4 in the otocyst, the those in Drosophila. Recently, it was shown that the Dqfortned dorsal root ganglia, some cranial ganglia, and the second regulatory element, which in the fly directs the transcription of branchial arch. SOHo- 7 expression in the otic pit and otocyst Deformed in posterior portions of the head segment,can, when is restricted to regions that will give rise to the nonsensory fusedto a transgene,direct the specificexpression of this trans- tissues of the inner . SOHo- 7 is not closely related to any gene in the hindbrain of a mouse (Awgulewitsch and Jacobs, identified vertebrate or Drosophila homeobox-containing 1992). Reciprocalexperiments have shown that human HoxD-4 . Since it is expressed in sensory-related structures can direct the expressionofendogenous Deformed in Drosophila and does not fit into existing classes of homeobox genes, (McGinnis et al., 1990) and that the HoxD-4 autoregulatory we propose the name SOHo- 7, for sensory organ homeobox-1 . element can direct head-specificexpression in Drosophila (Ma- [Key words: homeobox, sensory organs, retina, otocyst, licki et al., 1992). Targeted mutations in the homeobox gene anterior-posterior, development] Iloxa-I in mice, produced by homologous recombination in embryonic stem (ES) cells, revealed that it was required for the The highly complex morphogenetic movements, cell fate de- proper formation of the ear, cranial nerves, and hindbrain (Luf- cisions,and differentiation that occur during CNS development kin et al., 199 1; Chisakaet al., 1992). Recent experiments dem- are controlled by an intricate network of gene products, some onstrated that misexpressionofHoxd- I1 perturbs the positional of which are similar to those identified in invertebrates. For information in the limb, resultingin an apparent posteriortrans- example, in Drosophila, the homeobox geneshave been shown formation of the anterior portion of the limb (Morgan et al., to control basic patterning in the developing embryo through 1992). Such experiments indicate that homeobox genesplay key conversion of gradients of expression to a segmental pattern roles in patterning of the vertebrate embryo. (reviewed in Akam, 1987). The homeobox geneseve and ffz Approximately 30 years ago, it was predicted that cells in the have been proposedto be required for correct cell fate decisions retina had acquired positional information by virtue of their in neuronsin which they are expressed(Doe et al., 1987, 1988). locations relative to two orthogonal gradients, anterior-poste- In the eye, it has beendemonstrated that expressionof the rough rior (A-P) and dorsal-ventral (D-V) (Sperry, 1963). Heterotopic homeobox gene in photoreceptors R2 and R5 is necessaryfor transplantation of small groups of retinal cells in Xenopus in- proper development of the other photoreceptors (Tomlinson et dicated that they retained their A-P and D-V positional infor- al., 1988). More recently, the homeobox genesorthodenticleand mation when moved to different locations (Fraser, 1991). Since empty spiracles have been shown to be regulated by bicoid in a homeobox geneshave been shown to be involved in patterning manner similar to that of the gap genes(i.e., hunchback), sug- in other systems,we reasonedthat suchgenes could be involved gestingthat they specify broad regions in the Drosophila head in endowingpositional information in the retina. Thus, wesought to isolate homeobox-containinggenes from the retina and char- acterize their expression.In this article we describethe isolation Received Feb. 19, 1993; revised July 23, 1993; accepted July 29, 1993. of a novel homeobox gene from the embryonic chicken retina, We thank M. Mueckler for advice on cDNA libraries, H. G. Simon for expertise called SOHo-I, for sensory organ homeobox-1. In stage 14 on rn situ hybridization, E. Snyder for help in figure preparation. T. Burglin, D. Duboule, and C. Tabin for helpful discussions on homeobox genes, D. Noden for chicks [embryonic day 2 (E2)], it is expressedin the retina, helpful discussions on branchial arches, D. Contanche and B. Hay for expertise otocyst, prosencephalon,mesencephalon, and rhombencepha- and advice on anatomical identification, and N. Sucher, D. Wu, and L. Lillien lon. By E4, SOHo- is expressedat high levels in the nasal for critical reading of the manuscript. This work was supported by National Institutes of Health Grants T32 EY071 IO (D.L.D.), F32 EY06315 (D.M.F.), and (anterior) retina and at low levels in the temporal (posterior) R01 EY08064 (C.L.C.). retina, suggestingthat it may play a role in A-P patterning in Correspondence should be addressed to Connie Cepko, Department ofGenetics, Harvard Medical School. 200 Lonnwood Avenue. Boston. MA 02 115. the retina. High levels of expressionare also seenin the portions ‘Present address: Department orMolecular and Cellular Physiology, Stanford of the otocyst that give rise to non-neural structures and in the Medical School, Stanford, CA 94305. developing dorsal root ganglia(DRG) along the entire length of “Present address: Department of Biology, Boston College, Chestnut Hill, MA 02167. the spinal cord. The secondbranchial (hyoid) arch alsoexpresses Copyright 0 1994 Society for Neuroscience 0270-6474/94/140486-13$05.00/O high levels of SOHo-l on E4. The Journal of Neuroscience, February 1994, 14(2) 487

Materials and Methods essentially as described (Zeller and Rogers, I99 I) using “S-labeled RNA cDNA IrhrurJl construction and screening. Five micrograms of polyA + probes. Slides were pretreated with 2% aminoalkylsilane in dry acetone RNA were used to generate double-stranded oligo-dT-primed cDNA (Rentrop et al., 1986) and then 4% paraformaldehyde to retain sections as described (Gubler, 1988) except first-strand buffer contained 2.5 mM during subsequent steps. Six-micrometer paraffin parasagittal sections K + Blunt-ended cDNA was methylated with EcoRI methylase (New were hybridized with antisense RNA probe synthesized from EcoRI/ England Biolabs), tinkered with EcoRI linkers, and digested with EcoRI PvuII fragment (- 35 to 423) of SOHo- I after base hydrolyses. Paratilm (New England Biolabs). The cDNA was passed over a Cl00 column (American National Can) was used in place of coverslips during hy- (Pharmacia) and then size selected for cDNAs of > 1 kilobase (kb) by bridization. and 100 &ml of cold S-labeled RNA svnthesized from a agarose gel electrophoresis, glass purified (Vogelstein and Gillespie, 1979) Bluescript KS + PvuII Fragment (532-977) was included in the hybrid- cloned into phosphatased Xgtl 1 arms (Clonetech), and packaged with ization to block nonspecific hybridization. After washing, slides were Gigapack Gold (Stratagene), yielding a library of 6 x IOh recombinants; dipped in 1: 1 Kodak NBT-2 emulsion : water mixture and exposed for I x 10” plaques were screened with a synthetic I IO- (bp) 10 d at 4°C. Slides were counterstained with hematoxylin and eosin and oligonucleotide (Applied Biosystems) that was end labeled with y-“P- mounted with Dpx (Fluka). Sense probe from the same EcoRI/PvuII ATP (6000 Ci/mmol; New England Nuclear) by T4 polynucleotide ki- fragment was used to control for nonspecific hybridization. nase (Boehringer Mannheim Biochemicals). The I lo-mer sequence was Whole-mount in situ hybridization. A modification of the in situ based on a polymerase chain reaction (PCR) probe generated from first- whole-mount procedure ofConIan and Rossant (I 992) was used. Unless strand cDNA derived from E6 chicken eye. The PCR utilized Taq DNA stated, all PBT (PBS + 0.1% Tween 20) wash steps were done three polymerasc (Perkin-Elmer/Cetus) and consisted of 5 min at 95°C and times for 5 min each at room temperature. Stage 12-14 chick embryos then 35 cycles of 95°C for I min, 50°C for 2 min, and 72°C for 3 min were dissected, fixed in 4% paraformaldehyde for I hr at 4°C. washed under standard buffer conditions. The PCR primers were designed to in PBT, and dehydrated through 25%, 50%. 75% (methanol: PBT), then hybridize to conserved portions of murine homeobox genes. The 5’ 100% methanol. Embryos were rehydrated through a reverse methanol: primer was 5’ CTGGAGAAGGAATTCCAC 3’ and the 3’ primer was PBT series, washed in PBT. treated with 6% H,O, in PBT for 1 hr. and 5’ C/GCGATTCTGGAACCAGATCTT 3’. The resulting PCR product then rinsed in PBT. Embryos were treated with lb pg/ml proteinase K was subcloned into mpl8 and several clones were sequenced. The se- in PBT for 15 min. rinsed in PBT + 2 ma/ml elvcine. rinsed in PBT. quence of one of the clones (CK3) that contained a homeobox was used fixed in 0.2% glutaraldehyde/4% paraformaldeh;de in PBT for 20 min, to synthesize an oligonucleotide with the same sequence for screening rinsed in PBT, treated with 0.1% sodium borohydridc in PBT (mixed the cDNA library. Plaques were screened at a density of 150,000 per immediately before use), rinsed in PBT, treated with 0.25% acetic an- 24 cm x 24 cm plate, transferred to Hybond-N (Amersham), autoclaved hydride in 0.1 M triethanolamine-HCI, pH 8.0, for IO min. rinsed in 2 min dry cycle in an autoclave, UV cross-linked, and hybridized over- PBT, and then prehybridized for I hr in 50% formamide, 5 x SSC, pH night in 6 x- saline-sodium citrate (SSC) 0.05% sodium pyrophosphate, 4.5 (pH adjusted with citric acid), 50 fig/ml yeast RNA, 1% SDS, and 5 x Denhardt’s, 100 &ml salmon sperm DNA, and 0.5% SDS at 62°C 50 &ml heparin at 70°C. The digoxigenin-labeled RNA probe was with 2 x IO” cpm/ml. Filters were washed at 37°C in 4x SSC, 0.1% added to a concentration of 1 pg’ml, and then hybridization proceeded SDS for 1 hr, and then at 62°C in 2 x SSC, 0. I% SDS for 30 min. Filters overnight at 70°C. Embryos were washed in solution I (50% formamide, were exposed on Kodak XAR film with an intensifying screen at -80°C 5 x SSC, pH 4.5, 1% SDS) for 30 min at 7o”C, and then washed with for 2 d. After three rounds of plaque purification, two X clones, 5A-3 a I:1 mixture of solution I and solution 2 (0.5 M NaCI, 10 mM Tris- and 58-3, were subcloned into the EcoRI site of Bluescript KS+ (Stra- Cl, pH 7.5, 0. I% Tween 20) then three times with solution 2 for 5 min tagene). each. The embryos were then treated twice with 100 wcg/ml RNase (in Chick embryos. Fertilized White Leghorn eggs (SPAFAS, Inc., Nor- solution 2) for 30 min at 37°C and washed with solution 2 and solution wich, CT) were incubated at 38°C for indicated times or until the in- 3 (50% formamide, 2x SSC, pH 4.5) each for 30 min at 65°C. They dicated stages as defined by Hamburger and Hamilton (I 95 I). were then washed three times with TBST (0. I4 M NaCI. 3 mM KCI. 2.5 RN.1 isolation. RNA was prepared from embryonic tissues that were mM Tris-Cl, pH 7.5, and 0.1% Tween 26). Embryos were preblodked frozen in liquid N, and ground in a mortar and pestle, homogenized in with 10% sheep serum in TBST for I hr then incubated with anti- guanidinium thiocyanate. and prepared by the CsCl cushion method digoxigenin antibody (Ab) (precoupled to alkaline phosphatase; Boeh- (Chirgwin et al., 1979) or the acid-guanidinium-phenol-chloroform ringer Mannheim Biochemicals) at a dilution of 1:2500 in TBST and method (Chomczynski and Sacchi, 1987). This RNA was either used 1% sheep serum. Anti-digoxigenin antidbody (Ab) was preadsorbed on as total RNA or was polyA’ selected twice with oligo-dT cellulose acetone powder made from stage 12-l 5 chick embryos. Embryos were (Collaborative Research) as described by Kingston (1989). then washed three times in TBST for 5 min, five times in TBST for 1 Northern blots. An NcoI/XhoI fragment of SOllo- I (containing the hr each, and then three times in freshly prepared NTMT (100 mM NaCI, entire open reading frame) was gel purified and labeled by random 100 mM Tris-Cl, pH 9.5, 50 mM MgQ, 0.1% Tween 20, and 2 mM priming with a-“P-dCTP (50 @Ci) using the Amersham kit. This probe levamisole) for 10 min each. The embryos were then incubated in NTMT did not cross-hybridize to any other homeobox genes in Southern blots with 34 &ml nitroblue tetrazolium and I7 &ml 5-bromo-4-chloro- and similar results in Northems were obtained with probes containing 3-indolyl phosphate (Boehringer Mannheim Biochemicals) and devel- very little homeobox sequence (data not shown). RNA was electropho- oped until color was clearly visible. Embryos were washed in PBT and resed on a denaturing formaldehyde agarose gel and transferred in 20 x then 100 mM Tris-Cl, pH 8.0 and 10 mM EDTA. Embryos were pho- SSC to Hybond-N membrane (Amersham) by capillary action. Hybrid- tographed immediately or the next day to avoid increasing background. ization conditions are as described (Joyner et al., 1985) except hybrid- RNA probes. An EcoRI-PvuII fragment of SOHo- (-35 to 423) was ization temperature was 55°C. Blots were washed for I5 min at 25°C subcloned into the EcoRI-SmaI site of Bluescript KS (Stratagene). The in 2 x SSC, 0. I% SDS, and then at 65°C for 2 hr in 0.1 x SSC, 0.1% resulting plasmid was digested with Hind111 or XbaI, gel purified, and SDS. The blots were exposed to Kodak XRP film at -80°C for 2 d with isolated with glass powder/NaI (Vogelstein and Gillespie, 1979). HindIII- an intensifying screen. The Northern blot shown in Figure 2B (with digested plasmid was used for generation of antisense probes, and XbaI SOHo- probe) was exposed for 5 d with an intensifying screen. plasmids were used for the production of sense probes. “S-labeled RNA DNA sequencing. 5A-3 was sequenced on both strands in its entirety probes were prepared as described (Zeller and Rogers, 1991) and hy- with specific sequencing primers along its length with the Pharmacia drolyzed to approximately 150 nucleotides in length. Digoxigenin-la- T7 Sequencing Kit or the U.S. Biochemical Sequencing Kit. 5B-3 was beled RNA probes were synthesized from the same plasmids with the sequenced (on one strand) and it differed from 5A-3 only at its 3’ end, inclusion of 0.65 mM UTP/0.35 mM digoxigenin-labeled UTP (Boeh- in utilizing a different polyA site, and at its 5’ end, in terminating four ringer Mannheim Biochemicals) along with I mM ATP, CTP, and GTP. nucleotides short of its presumed initiation ATG. Homology between Synthesis proceeded for 2 hr at 40°C. The reaction was then digested the I IO bp oligonucleotide probe (used to screen the cDNA library) and with DNase I (RNase free) (Boehringer Mannheim Biochemicals) for clones 5A-3 and 5B-3 only consisted of a 20 bp region of homology 15 min at 37”C, ethanol precipitated, rinsed in 70% ethanol, and re- from the 3’ of the probe. A different homeobox gene than the one suspended at a concentration of approximately 0. I rg/ml. An aliquot encoded by the PCR probe was thus isolated from the cDNA library. of the RNA product, prior to the DNase I digestion, was run on an Compressions were resolved by subcloning portions of 5A-3 into mp18 agarose gel to estimate amount of RNA and efficiency of transcription. or mp I9 and employing deazaguanosine or inosine nucleotides (Phar- Three-dimensional reconstruction of in situ hybridization sections. macia). Sequences were aligned using the GCG Wisconsin package. Camera lucida drawings ofthe in situ hybridized sections were prepared Radioactive in situ hybridization. In situ hybridization was performed from dark-field images through a 10x objective on a Zeiss Axiophot 488 Deitcheretal.*Sensoty Organ Homeobox Gene

-35 TCCGCCGCCACTCCTGCGGTTAGGCTCAGGCTCAGCGCGCC

ATG GTG CAG CTC GGG GGA GGC CGC GGA GCC CCA CCG CCT CTC CTG GCC CCA CCG TCG GCC Met val gln leu gly gly gly arg gly ala pro pro pro leu leu ala pro pro ser ala

TTC AGC ATC GAC AGC ATC CTG CAG CCC GGT CCC CGC TGC CAG GCC CGG GAG CAG GGG AGG phe ser ile asp ser ile leu gln pro gly pro arg cys gln ala arg glu gln gly arg

GCC CGC TGC GCG CTG CCG GAG GAC GAG GAG GAG GAG GAG GAG GAA GAA GAG GGG CCT GCG ala arg cys ala leu pro &l asn alu alu alu alu alu alu alu alu alu gly pro ala

181 GAG GAA CAC CCC ACT AAA GGC TCC ACC GAC TCG GGC AGC GAG AGG CTG CTG GCG GAA GGG 61 glu glu his pro thr lys gly ser thr asp ser gly ser glu arg leu leu ala glu gly

241 CCG CGC CGC GCG GAT GCC GAG GCC GAA GGC GCG GTT TCA CCG CTC TCC ACG GAG AGG TTC 81 pro arg arg ala asp ala glu ala glu gly ala val ser pro leu ser thr glu arg phe

301 CGC GGA TGC CGA CAG CCG TCG CTG CGG GAT ACC GGG GGC TGC GGT AGA GAG AGC GGC AGG 101 arg gly cys arg gln pro ser leu arg asp thr gly gly cys gly arg glu ser gly arg

361 TGT TCA GCG GCG GGA GGC AAG AAG AAG ACG CGG ACC ATC TTC TCC AAG AGC CAG GTC TTC 121 cys ser ala ala gly gly fis lvs lvs thr ara thr lie Dhe ser lvs sw aln val the

421 CAG CTG GAG TCC ACC TTC GAC GTG AAG CGC TAC CTG AGC AGC GCC GAG CGG GCC GGT CTG 141 sin leu alu ser thr ohe asp val lvs ara tvr leu ser ser ala olu ara ala alv leu

481 GCC GCC GCG CTG CAC CTC ACC GAG ACG CAG GTG AAG ATC TGG TTC CAG AAC CGC CGC AAC 161 dla ala ala leu his leu thr alu thr aln val 1~s ile Luz Dhe aln asn ara ara a.zn

541 AAG CTC AAG AGA CAG CTG TCG GCT GAA CCC GAG GGT CCG GGC CAA GCG GAA CCC CCA GGG 181 Ivs leu lvs ara aln leu ser ala glu pro glu gly pro gly gln ala glu pro pro gly

601 GAG CCT CCT CCG CCT CCC GCC GCC TCT TTC TCC TTC CCG TCC CTA TAC AAG GAC AGC GCC 201 glu pro pro pro pro pro ala ala ser phe ser phe pro ser leu tyr lys asp ser ala

661 CTG TTC AGC CGC TGC CTG CTG CCA CTC CCC TTT CCT CTG TTC TAC CCG GGC AGC GCC ATC 221 leu phe ser arg cys leu leu pro leu pro phe pro leu phe tyr pro gly ser ala ile Figure 1. Nucleotide and deduced amino acid (aa) sequence ofthe SOHo-l 721 CCC TAC CTC TGC CTT CCC GGT CCG GTC AAG CAC TTC AGC CTG CTG GAC GGG GAC GTA TAG gene and comparison to 241 pro tyr leu cys leu pro gly pro val lys his phe.ser leu leu asp gly asp val * from TgHbox.5 and ceh-9. A, The se- quence of SOHo-I. Both the first nu- CGTCTCACCTCGGCCCCCGCCCTGTCTCTTGCAGGGGGCCGATGGTCCCTGTGGCACAGCCGTACCACCATCAGCTTTC cleotide and the initiation methionine GCCCCGACACGCGGCAGCCGCTCCCAGGCCCGGGGTCCCCCACACGCTGCCGTGTGCCGTCGTGTCAGAGGAGCTTCAA CGAGCGCTGCACTCTGCTCGAGACGGAGAAGAACGGAGCTGCCAAAGAAACCGGGATGCGCTGTGTCTCCTTTCCCTCC are designated position 1. Upstream in- ACAGCCTTTCCGTGGCCGCAGTTTAGGAAAGATGATATCTGCCTCAGTCCACGGGAATGGTGCCATCGCGGCTGCCCCC frame tFanslationa1 stop codon at - 15 GCACCGTACCGCCACCCGAGAGCGTTGGGTACAGCCCCATCCACTCCGTACGCTCCTCTATCTCACATTCTCCGCCTTT is indicated in italics. The acidic-rich AGTACACCTTCAATTAACGCTTCCCCATCTCTGACACTATCAAAATGCAGACACCTCTCTAAATCTCTGTAGCTTCTCT region is underlined (aa 47-57) and the GAATTTTGAATTTTCGAACCAAACAAGTCCTCTGTCACACTTTGTGCTAACCGGTTCCCAAATCAGGGCCCGTCTGATG hgmeodomain is both underlkzed and TTTGGCGCACAAGTTACTCATGCTCCTTACGCTAACCCCGCCCCCGGTTGCTCAGGGGATTATTTTATCTGCTGCCCTA boldface faa 127-186). The four outa- CTGTAACGAAGTGCTTTCTTTTGTTTTTTGTTTTAATCCCCCTTAAATCTCCAACCACATTTGTCTTCTTTTGGTTTCC tive”polyk sites (AATAAA) are under- GAGTTCACACGCTTjATATTACAAATTGGGAAATAAAACTTTTCTATCAGAAACCCGAGCTGCATGCCTGTTCCGTACA lined at the 3’ end. B, Alignment of GCCGCTCCAGGGCAGTGTCCAAAGAAAGACGTTTCCTAGAGTGCATCGACGGGTGTTTGTTTTAAAAATAA~GGTTGAA SOHo- homeodomain with those from CGTGCAATCGTGGTGTGGCGAAATTCAGAAGTGCGTGCTAAAATGGGAGACTTGTTGAGTTTTCCGGAGGGAAAAAAAA TgHbox5 of sea urchin and ceh-9 of C. AATAATAAAATGAAAAAAAAAATAAAGAAAAGAAAAAGAGAAAAAAAAAAAAAAAA elegans. Amino acids that are con- served between SOHo- I and TgHbox5 1 10 20 30 40 50 60 and ceh-9 are indicated by the reverse SOHo- boxedregions. The single-letter code for TgHbox5 amino acids is used. ceh-9 microscope. Grain density considerably above background was subjec- 1986) and has an in-frame stop codon located 15 nucleotides tively judged to be positive for expression. Semiserial sections were upstream. Somewhat unusual in this sequenceis the entered into a Sun 386i workstation with a digitizing tablet. Sections were reconstructed with the Computer Aided Reconstruction Package position of 11 continuous acidic amino acids (47-57) relative (CARP; Biographies, Inc., Dallas, TX). Three-dimensionally recon- to the homeodomain; typically such acidic stretchesare found structed otocysts were displayed on a high-resolution color monitor and on the carboxyl side of the homeodomain instead of the amino then photographed. side (Falzon et al., 1987; Kessel et al., 1987; Simeone et al., 1987; Wright et al., 1987). Results Isolation qfa novel chicken homeoboxgene Homology to other homeobox-containinggenes, TgHboxS and We isolatedand identified a novel homeobox gene from an E8 ceh-9 chicken eye cDNA library with a PCR-generatedprobe. The The homeobox in the isolated cDNA does not appear to be a predicted protein is composedof 259 amino acids, including a member of the four identified vertebrate Hox clusters since it homeodomain from amino acid 127 to 186 (Fig. 1A). The pro- is not a paralog or homolog of any identified vertebrate Hox- posedinitiation methionine (labeled position 1) was chosenas classhomeobox geneand it is not very related to any Drosophila suchsince it conforms to the Kozak consensussequence (Kozak, Antennapedia-class homeobox genes, which define Hox-class The Journal of Neuroscience, February 1994, 14(2) 499

A B C

kb 1 2 3 4 5 1 2 3 4 5 6 7 6 9 10 1 2 3 9.5- 7.5-

4.4’

2.4-

(1 actin -

Figure 2. Expressionof SOHo- in the embryonicchicken eye and in variousembryonic tissues by Northernblot analysis.A, Expressionin the eye. Eachlane contained 10 pg of total RNA from chickeneye at the indicatedembryonic times. Size markers (Bethesda Research Labs) are shown to the leji in kb. Lane 1. embryonicday 3.5-t (E3.5-E4);lane 2, E6; lane3, E8; lane 4, E9.5;lane 5, E13.Below, blot wasreprobed with an EcoRI fragmentof the humancu-actin gene to control for amountand integrity of RNA. B, Expressionof SOHo- in embryonictissues. Each lane contains 5 ~a of noIvA+ RNA from indicatedembryonic tissues. In order to seeexpression in embryonictissues other than the eye, the eyeswere not inchidedin -anyof the samplesexcept lane 1: Lane I, E6 eye; lane 2. E4 whole embryo; lane 3, E4.5 head, lane 4, E4.5 torso;.lane 5, I?8 head, lane 6, E8 torso;lane 7. E20 brain;lane 8, E20 heart;lane 9, E20 intestine;lane 10, E20 liver. Below, blot wasreprobed with an EcoRI fragmentof the humana-actin gene to control for amountand integrity of RNA. C, Expressionof SOHo- in developingsensory organs. Each lane contains 5 ~g of total RNA from the indicatedtissues. Lane I, E6 eye; lane 2, E7 DRG, lane 3, ESotocyst. Amount and integrity of RNA werecontrolled for by stainingduplicate lanes with ethidiumbromide.

homeobox genesin vertebrates. Moreover, this homeobox gene RNA from whole embryonic eyes. At E3.5-E4, strongly hy- is not closely related to any other homeobox genesfrom Dro- bridizing bands of 1.8 and 2.0 kb were observed (Fig. 2A, lane sophilacharacterized to date. Since it is expressedin the retina, 1). A steady decline in expressionwas evident in RNA from E6 the otocyst, and DRG, which are all sensoryorgans or sensory- and E8 eyes (Fig. 2A, lanes 2, 3), which then reached a lower related structures (seebelow), and does not clearly fit into ex- steady state at E9.5 and El 3 (Fig. 2A, lanes 4, 5). SOHo- is isting homeobox classes,we propose the name SOHo-1, for also expressedin adult chicken retina (data not shown).Limited sensory organ homeobox-I. The homeodomain of SOHo-1, material prevented determination of the onset of SOHo- ex- however, doeshave striking homology to the homeodomain of pression by Northern analysis prior to E4 in the eye. From the seaurchin geneTgHboxS (Wang et al., 1990); 53 of 60 (88%) partial sequencingof another clone, it appearsthat the presence amino acids are identical. SOHo- and TgHbox.5 also sharethe of two transcripts is due to utilization of different polyA sites; unusual positioning of the acidic region relative to the homeo- four such sites were found in the 3’ untranslated region (Fig. box. The homeodomainsof SOHo- and ceh-9 (Hawkins and 1A). Northern blots probed with 3’ untranslated sequencespe- McGhee, 1990) from Caenorhabditiselegans are also similar, cific to the larger cDNA clone hybridized to the 2.0 kb but not with 60% identity (seeFig. 1B). From amino acid homology, it to the 1.8 kb transcript, further supporting the notion that the would appearthat thesethree genesare membersofa new family transcripts differ in size due to 3’ untranslated sequencesre- of homeobox genes.We suggestthat this new family be named sulting from differential use of polyA sites(data not shown). the NEC class, for nematode echinoderm chicken-the organ- Five micrograms of polyA+ RNA were prepared at various isms from which they were isolated. The significant homology time points and from different portions of embryos for Northern between SOHo-I, ceh-9, and TgHboxS in such evolutionarily analysisto seeif any other tissuesof the embryo contained the diverse creatures makes it likely that other members of the SOHo- transcript. RNA from E6 embryonic eyes was run for proposedNEC family will be found in many other groups of comparison purposes(Fig. 2B, lane 1); although overexposed animals including mammalsand insects.Whether SOHo- I and in Figure 2B and thus difficult to discern, the 1.8 and 2.0 kb TgHbox.5 representtrue homologs or different membersof the transcripts were observed with shorter exposure times. E4 em- samefamily will becomeclearer as more related genesare iden- bryos with the eyes removed had low but detectablelevels (Fig. tified. 2B, lane 2). On E4.5-E5, expressionwas observed in both the head and torso (seeFig. 2B, lanes3,4), and on E8 head minus SOHo- 1 is expressedduring eye showed moderate levels of expression while much lower In order to characterize the expressionof SOHo- I during chick- levels were detectable in the torso fraction (Fig. 2B, lanes5, 6). en , Northern analysiswas performed on total No expressionwas detected from brain, liver, heart, and intes- 490 Deitcher et al. * Sensory Organ Homeobox Gene

Figure 3. Expression pattern of SOHo- in stage14.5 (approximately E2)chick embryo: Whole-mount in situ hybridization of stage14.5 chick hy- bridized with antisense(A) and sense (B) probesof SOHo- (lateral views). Dark purple regions indicate areasof specifichybridization with the antis- enseprobe. Note intensesignal in the (oc) from whichthe retinawill form and the semicircularring on the sideof the embryo,which is the lateral posteriorportion ofthe otic pit (op), the inner ear anlage.Signal is alsoseen in the brainanlage in the prosencephalon, mesencephalon,and the rhombenceph- alon.Magnification, 27 x .

tine from E20 embryos (Fig. 2B, lanes7-10). In all samplesthat probed with control senseRNA varied from almost no back- testedpositive for the SOHo- I transcript, the predominant tran- ground to a faint bluish background in the head, but the level scripts were 1.8 and 2.0 kb, the same molecular weights as the of blue was always much lessintense than the signal observed transcripts seenin the eye. in antisenseembryos (Fig. 3B). While the whole-mount method Northern analysiswas also performed using 5 Kgof total RNA was very useful for stainingstage 12-l 4.5 embryos, its usefulness from eye, DRG, and otocyst to seeif SOHo- I was expressedin for older embryos was limited as control senseprobes revealed sensorystructures. RNA from E6 eye had the highest level of that nonspecificbackground increaseddramatically. In order to expression(Fig. 2C, lane l), but strong expressionwas seenin localize SOHo- transcripts in older embryos, ?S-labeled RNA the ES otocyst as well (Fig. 2C, lane 3). Faint but detectable probes were used on paraffin sectionsof different stagedem- levels of SOHo- transcript were seenin E7 DRG RNA (Fig. bryos. 2C, lane 2). In all three samples,transcripts of 1.8 and 2.0 kb were observed. SOHo- expression in retina Radioactive in situ hybridization was performed on sectionsto SOHo- 1 spatial expression in early development localize the expressionof SOHo- I in the retina. In the stage23 Whole-mount in situ hybridization was performed to determine (E4) chick, the highest domain of SOHo- expressionwas al- whether SOHo- I was expressedin early stagesof development. most entirely in the nasalretina with a minor portion extending Digoxigenin-labeledantisense and senseRNA probeswere pre- across the ventral furrow into the extreme temporal-ventral pared and hybridized to a number of chick embryos between retina. Except for this small patch of temporal-ventral expres- stages12 and 14.5 (Hamburger and Hamilton, 1951). In Figure sion, SOHo- was expressedat markedly lower levels on the 3, a typical stage14.5 embryo is shown after stainingfor alkaline temporal side. This pattern of hybridization was evident in all phosphatase,the tag usedto detect the presenceof the SOHo- the parasagittal sections along the medial-lateral axis (Fig. transcript. Prominent staining was observed in the optic cup 4B,DJ’,H). Reproducible hybridization was also seenover the (Fig. 3A, labeledoc), both in the inner layer of neuroepithelium developing lens (Fig. 4F’,H). The expressionof SOHo- ex- which gives rise to the retina and in the outer layer of the optic tended from the retina into the , the neuroepithelial- cup which gives rise to pigmented epithelium. Intense, asym- derived structure that later supports the formation of the optic metrically distributed staining was also observed in the otic pit nerve (Fig. 5D and F) and which possessessome activity in of stage 14.5 embryo (Fig. 3A, labeled op). The most intense directing the paths of retinal axons (Harris, 1989). In postnatal staining in the otic pit was in the region most distant from the animals, SOHo- is expressedin all three layers of the mature hindbrain. Strong staining was also present in the , retina-the outer nuclear, inner nuclear, and ganglion cell layers starting at the most anterior portion of the telencephalon and (Fig. 5B). extending caudally to the posterior end of the rhombencephalon (Fig. 3A). Expression of SOHo- was seenin the SOHo- 1 expression in the otocyst of a stage12 chick (data not shown). The whole-mount view of The vertebrate inner ear developsfrom an epithelial thickening, the optic cup did not reproducibly reveal asymmetric expression called the , on the surfaceof the head adjacent to of SOHo- I in the stage14.5 retina. The purple precipitate that the hindbrain. The otic placodeinvaginates to form the otic pit, resulted from the whole-mount in situ procedure was difficult which subsequentlypinches off to form the (or oto- to detect after sectioning,so it could not be determined whether cyst). Ultimately the otic pit gives rise to all of the membranous SOHo- was expressedasymmetrically at stage 14.5. Embryos components of the inner ear, including the sensoryorgans for The Journal of Neuroscience, February 1994, f4(2) 491

Figure 4. Expression pattern of SOHo- in stage 23 chick eye by in situ hybridization: Bright-field and respec- tive dark-field parasagittal sections of the eye. Panels are arranged from me- dial (top) to lateral (bottom). The ori- entation of all the sections is indicated in A: nasal (anterior) (IV). temporal (posterior) (T), dorsal (D), and ventral (V’). Retina (r), pigmented epithelium (pe), and lens (I) are indicated. Arrow marks position of the ventral furrow. Scale bar, 100 am. 492 Deitcher et al. l Sensory Organ Homeobox Gene

Figure 5. Expression pattern of SOHo- in posthatch day 0 (PO) chick retina and stage 23 optic stalk by in situ hybridization. Outer nuclear layer (onl), inner nuclear layer (id), ganglion cell layer (gel), and optic stalk (OS) are in- dicated. A, Bright-field photograph of cross section through PO chick retina; B, same section shown in dark field. C, Bright-field photograph of parasagittal section through the optic stalk, D. same section shown in dark field. E, Bright- field photograph of oblique parasagittal section through optic stalk, F, same sec- tion shown in dark field. Scale bar, 100 urn.

hearing and balance, and the associatedneurons of the VIIIth The expressionof SOHo- I was studied at three time points cranial ganglion. In the chick, the otic pit is formed on E2. At during the course of sensoryorganogenesis: E2, E4 (stage23), this sametime, neural progenitor cells bud off the ventromedial and E4.5 (stage 26). On E2 the otic pit showedan asymmetric wall to form the VIIIth cranial ganglion. The otocyst is formed distribution of SOHo-I, with the most intense staining in the by E3 (stage 18). From E3 to E6.5 (stage 30), evagination and posterolateral portion of the optic pit (Fig. 3). differential growth of the walls of the otocyst serve to subdivide At stage23, high SOHo- expressionis still primarily a con- the inner ear into all of its component parts (semicircular canals, tiguous patch, with highestlevels in the lateral third of the otic utricle, saccule,basilar papilla, and lagena). Separation of the vesicle and continuing posteroventrally in the middle third of sensoryorgans into eight discretecomponents occurs gradually the otic vesicle (Fig. 6). A secondregion of high expressionis (Knowlton, 1967). What is initially a contiguous patch of cells found at the junction of the primordial endolymphatic duct in on the anteroventromedial region of the otic pit on E2.5 has some sections(data not shown). The endolymphatic duct is a subdivided into three main sensoryregions by E4 (stage24): (1) nonsensory structure that will eventually connect the inner ear an anteroventral area that gives rise to the sensory organs of fluid system to the brain ventricular system. Some cells in the the utricle and the anterior and lateral semicircular canals, (2) adjacent VIIIth cranial ganglion are also expressingdetectable a posteroventrolateral region that givesrise to the sensoryorgans amounts of SOHo- I (data not shown). of the posterior semicircular canal and the macula neglecta,and By stage26, the spatial distribution of SOHo- in the otic (3) a ventromedial area that gives rise to the sensoryorgans of epithelium hasbecome more complex and patchy (Fig. 7). Three- the sacculeand the enlarging primitive lagena. The primitive dimensionalreconstruction of the expressionpattern from serial lagenafurther separatesinto two regions:the definitive lagena sectionswas helpful in recognizing the significanceof the patchy (a vestibular organ) and the basilar papilla (the auditory organ). distribution (Fig. 8). SOHo- appearsto be high in regionsthat The final pattern of eight discrete sensoryorgans is apparent by will not form sensoryorgans. That is, expressionis absentin an E6.5 (stage 30). The majority of the hair cells and supporting anteroventral patch, a posteroventrolateral patch, and two sep- cells in the basilar papilla undergo their final mitotic division arate patcheson the ventromedial surface that may represent between E5 and E8 (Katayama and Corwin, 1989). The gen- the forming sensoryorgans in the sacculeand lagena (Fig. 8). eration of the vestibular hair cells probably precedesthat of the By stage26, SOHo- is also uniformly high on the primordial auditory hair cells by approximately l-l .5 d, basedon the initial endolymphatic duct. Expression is still evident in the Vth and expression of a hair cell marker (Bartolami et al., 1991) and VIIIth cranial ganglion (Fig. 7&D), although the intensity of comparisonswith cell generation in the mouseinner ear (Ruben, hybridization in the VIIIth cranial ganglion appearsto vary in 1967). different regions,and is not particularly high in Figure 7. The Journal of Neuroscience, February 1994, M(2) 493

Figure 6. Expression pattern of SOHo- in otocyst and hyoid arch at stage 23 by in situ hybridization: par- asagittal section (labeled as follows: otocyst, o; endolymphatic duct, arrow- heaa; and second branchial arch, 2. An- terior (A). nosterior (P). dorsal (D). and ventral (k’j are as indicated. A.‘ Bright- field photograph of stage 23 embryo through otocyst and second branchial arch (lateral portion); B, same section shown in dark field. Scalebar, 100 pm.

expression is mainly expressed in the developing nervous sys- SOHo- 1 expression m the second branchial arch and tem, the expression patterns and functions of ceh-9 and Tg- frontonasal process Hbox5 have not been determined. At stage 23, in situ hybridization revealed that the apical tip of the second branchial (or hyoid) arch expressed high levels of Expression of SOHo- in the brain anlage SOHo- transcript in mesenchymal cells (Fig. 6B). By stage 26, Early expression of SOHo- in the anterior CNS can be found SOHo- I expression has expanded to encompass the entire sec- throughout the brain anlage. The brain of stage 14.5 chick con- ond arch (Fig. 7F) except the gap in the middle, which may sists of the prosencephalon, the mesencephalon, and the rhomb- correspond to the superior cervical sinus. Most of the mesen- encephalon: three swellings at the anterior end of the neural chymal cells of the second arch are derived, having tube. The timing of early SOHo- I expression in the chick brain migrated from the fourth (Lumsden et al., 199 1). approximately coincides with the expression of Emx- I and Emx- The hyoid arch in chick gives rise to portions of the columella 2, the mouse homologs of the Drosophila homeobox gene empty auris, the middle ear bone of chicks, and part of the hypobran- spiracles, and Otxl and Otx2 (Simeone et al., 1992a,b), the chial skeleton, which supports the tongue (Romanoff, 1960). At mouse homologs of the Drosophila homeobox gene orthoden- stage 23, a small but intense domain ofexpression was observed title. Like these genes, SOHo- has a clearly defined posterior in the mesenchyme of a restricted portion of the frontonasal expression boundary in the early neural tube. SOHo- extends process (data not shown). This region will ultimately generate farther caudally than Otx and Emx genes, overlapping in the tissue of the forehead and dorsal beak. hindbrain with members of the Hox-2 cluster. The mouse ho- mologs of the gene Distal-less, Dlx (Price et al., 199 1) and TES- I SOHo- expression in the DRG (Porteus et al., 199 l), are also present in the developing anterior The DRG, which at stage 26 are visible as periodic condensa- portions of the brain at slightly later embryonic times. Since tions of cells along the length of the spinal cord, clearly hybrid- SOHo- expression includes the entire brain anlage by stage ized to the antisense probe of SOHo- (Fig. 9B). DRG along 14.5, it could act in conjunction with chick homologs of Emx- the entire length of the spinal cord expressed the SOHo- gene 1, Emx-2, Dlx, Otxl, Otx2. and TES-1 in specifying portions at apparently equal levels. Unlike the earlier pattern at stage of the anterior neural tube and with the Hox class of homeobox 14.5 in which SOHo- was restricted to the anterior portion of genes in specifying portions of the hindbrain. At later devel- the embryo, the pattern in the DRG extended to the caudal opmental times (E4), SOHo- expression was seen at above portions of the spinal cord. background levels in cells lining the brain ventricles, but the level was not comparable to that seen in retina, otocyst, or DRG Discussion (data not shown). We have cloned a novel homeobox gene and characterized its complex distribution in the embryonic chick Expression of SOHo- in the eye during the early stages of neurogenesis. Its lack of homology to The vertebrate eye forms from an evagination of the neural other identified mammalian and Drosophila homeobox genes tube, the optic vesicle. Shortly thereafter, at stage 13, the optic and its striking homology to the sea urchin gene TgHbox5 and vesicle invaginates to form the optic cup. The optic cup is com- the C. elegans gene ceh-9 suggest that these genes represent a posed of an inner layer that will give rise to retina and an outer new class of homeobox genes. It is likely that homologs of layer that will form the pigmented epithelium, a non-neuronal SOHo- will be identified in other organisms. While SOHo- supporting tissue. SOHo- is expressed in what appears to be 494 Deitcher et al. - Sensory Organ Homeobox Gene

Figure 7. Expression pattern of SOHo- in otocyst and hyoid arch at stage 26 by in situ hybridization. The A-P orientation of these sections are reversed relative to sections in Figure 6. All sections are parasagittal, and relevant embryonic structures are labeled as follows: ootocyst, 0; Vth cranial ganglion, V; VIIIth cranial ganglion, VIII; and second branchial arch, 2. Anterior (A), posterior (P), dorsal (D), and ventral (V) are as indicated. A, Bright-field photograph of stage 26 embryo through otocyst (lateral portion); B, same section shown in dark field. C, Bright-field photograph of stage 26 embryo through otocyst but more medial than A and B; D, same section shown in dark field; E, Bright-field photograph of stage 26 embryo through hyoid arch; F, same section shown in dark field. Scale bar, 100 pm. the retinal anlage (inner layer) and perhapsthe outer layer as poral) asymmetry in the retina, as reflected in ganglion cell well. By stage23 (-E3.5) when most of the retinal cell types targeting, doesnot becomeapparent until much later, after ax- are being generated, the lens is well developed, and the tissue ons emergefrom the retina and segregateaccording to their A-P giving rise to the pigmented epithelium is starting to become position in the retina at approximately E6. While some retinal pigmented, the asymmetric expression ofSOHo-I along the A-P ablations suggestedthat this axis is fixed in the chick between axis of the retina is striking. Anterior (nasal)-posterior (tem- E3 and E4 (de Long and Coulombre, 1965),other ablation stud- The Journal of Neuroscience, February 1994, 14(2) 495

r AV PVL’

D D t p-f l-- p

Fgure 8. Three-dimensionalreconstruction of expressionpattern of SOHo- in otocystat stage26. The otocystis shownfrom both a lateral (/efl) and a medial(right) view. Positive expressiondomains are indicatedin yellowand negativeexpression domains are indicatedin pink. Developingsensory patches are negative,including an anteroventralpatch (A V), a posteroventralpatch (PW), the saccularmacula (.S), and the primitive lagena(L). The endolymphaticduct (E) ispositive, as are the developingsemicircular canals. Orientation is as shown: dorsal, D; posterior, P. A seriesof medialsections were used as sense controls; these are indicatedas blue broken lines, sincetheir SOHo- expressionlevels cannot he assessedfor this specimen. ies indicated that this occurs between stages12 and 13 (-E2) anterior axons stained poorly; the spatial expressionbetween (Crosslandet al., 1974). Early determination of the A-P axis of E3 and E6 was not described. The presenceof TRAP on de- the chick retina is consistent with experiments in Xenopus in veloping ganglion cell axons, and the absenceof TRAP on pro- which small groupsof undifferentiated retinal cells were labeled genitor cells, where patterning may take place (Fraser, 1991) and transplantedto other locations in the retina. The descendent may suggestthat it plays a role in axon guidance. The earlier retinal ganglion cells acted as though they retained their original expression of SOHo- at E2 and its homology to homeobox positional value in that the axons grew to the original tectal genesmay suggestthat it plays an important role in either spec- target area (Fraser, 199 1). ifying or reflecting the A-P patterning of the retina. However, Recently, two studies employed monoclonal antibodies to due to limitations of the whole-mount in situ protocol, the first label that were preferentially distributed in the tem- appearanceof the asymmetric distribution of SOHo- has not poral (posterior) retina in chick (Trisler, 1990; McLoon, 1991). yet been established.If SOHo- is involved in A-P patterning One of the monoclonal antibodies recognized the cell surface ofthe retina, then the patch ofexpression that crossesthe ventral protein TOP,,, which was preferentially localized in the pos- furrow may indicate that the developmentally defined anterior terior retina in a gradedfashion throughout most of embryonic compartment of the retina may not directly correspond to the development (E4-E18) (Trisler, 1990). A reciprocal pattern was anatomical one defined by the furrow. seenin the chick tectum, suggestingthat TOP,, may play a role Several molecules have been shown to have an asymmetric in axonal targeting. The other cell surface protein, temporal or graded distribution in the retinal D-V and A-P axes (Trisler retina axon protein (TRAP) (McLoon, 199 l), was first detected et al., 1981; Constantine-Paton et al., 1986; Trisler and Collins, at the end of E3, on a small patch of ganglion cell axons dorsal 1987; Nomes et al., 1990; Trisler, 1990; McCafferty et al., 1991; to the optic stalk, but its distribution was not asymmetric with McLoon, 1991; Monaghan et al., 1991). Of particular interest, respect to the A-P (nasal-temporal) axis. By E6, the differen- with respectto establishingthe retinal D-V axis, are Pax2 and tiating posterior ganglion cell axons stained heavily while the aldehyde dehydrogenase(AHD-2). Messagefor Pax2, a homeo- 496 Deitcher et al. l Sensory Organ Homeobox Gene

reflects transcriptional activation after the cells have migrated from the otic pit and associatedto form a ganglion. Recently, three homeobox genes(dlx3, msh-4 and msh-C) were found to be expressedin a spatially restricted pattern in the developing zebrafish ear (Ekker et al., 1992). The dfx3 gene is first expressedin a group of ectodermal cells prior to their organization into the otic placode. We have not examined whether SOHo- is expressedat an equivalent stagein the chick. Later in zebrafish development, at 16 hr, approximately the equivalent of stage14.5 in the chick, the dlx-3 geneis expressed in the dorsal posterior region of the otic placodein what would be an adjacent and nonoverlapping region of the otic placode with respectto SOHo- expression.By 24 hr, dlx-3 is expressed in the dorsomedial portion of the otocyst and msh-D was ex- presseddorsally in a portion of the otocyst that is central with respect to the lateral medial axis. If, as suggestedby Ekker et al. (1992) dlx3 and msh-D expressionare affected by inductive signalssuch as int-2 (Repressaet al., 1991) then the adjacent and nonoverlapping domain of SOHo- I expressionmay restrict which areas of the otocyst are capable of respondingto these signals.Alternatively, the domain of SOHo- expressionmay reflect its location relative to the hindbrain or someother struc- ture. For example, since the SOHo- domain is most distant from the hindbrain, its expressionmay be selectively downregu- lated by a hypothetical signal originating from the hindbrain, thereby explaining the spatial pattern of expressionin the oto- cyst. Such a model should be testable by experimental manip- ulation. Later, msh-D and msh-C are expressedin macular pre- cursor cells that give rise to the hair cells. These cells do not expressSOHo- in the chick. SOHo- expressionin the DRG Figure 9. Expression pattern ofSOHo-I in dorsal root ganglion (DRG) by in situ hybridization: medial parasagittal section through stage 26 The expressionof SOHo- in the developing DRG wasapparent embryo to reveal SOHo- expression in the DRG. A, Bright-field pho- by stage23 and strongly positive at stage26. The neural crest tograph of stage 26 embryo through the DRG; B, same section shown cells that give rise to the DRG migrate from the neural tube in dark field. Scale bar, 100 pm. and first appearas recognizableganglionic masseson both sides of the spinal cord between stages18 and 19. and differentiated bipolar neuronswith large cell bodies commingle domain gene with a paired box, is preferentially expressedin in the DRG until E8-ElO, when differentiated and undifferen- the ventral retina (Nornes et al., 1990). AHD-2, an enzyme tiated cells segregateinto the ventrolateral and dorsomedial capable of producing retinoic acid from retinaldehyde, is ex- regions of the DRG, respectively. Between ES and E8 neuro- pressedin the dorsal retina (McCafferty et al., 1991) although blasts differentiate and send processesto the spinal cord and recent evidence indicatesthat ventral retina can synthesizemore the dermis (Levi-Montalcini and Levi, 1943). While relatively retinoic acid than dorsal retina (McCafferty et al., 1992). Pux2, high expressionis seenin the mixed population of neuroblasts AHD-2, TRAP, and SOHo- are all asymmetrically distributed and differentiated neurons in the DRG at stage26 (E4.5), de- in the retina; each molecule is distributed with more on one tectable but low expressionwas seenby Northern analysis at half of the retina than on the other half. The distribution of E7. It is therefore possible that SOHo- expression is extin- these molecules suggeststhat the retina may be organized in guished as neuroblastsdifferentiate into mature neurons. compartments of nasal, temporal, dorsal, and ventral. Com- partment-basedmolecules, in conjunction with as yet undeter- SOHo- expressionin the secondbranchial arch and cranial mined gradient molecules,may specify retinal position, enabling ganglia precisetopographical connections to be formed. The second branchial (hyoid) arch expresseshigh levels of SOHo- by stage26, as do the Vth and VIIIth cranial ganglia. Expression of SOHo- in the ear The hyoid arch is mainly derived from neural crest cells of By stage14.5, the otic placodehas invaginated to form the otic rhombomere 4 (Guthrie and Lumsden, 1991). Neurons of the pit but has not yet completed closure to form the otic vesicle. Vth cranial ganglia are derived both from ectodermal placode At this stage, the expression of SOHo- delineated the non- and from the neural crest of rhombomere 2, while the neurons neurogenicregion of the otic pit on the lateral posterior half. of the VIIIth cranial gangliaare derived from the otic placode. The negative regions are fated to becomesensory end organsas The expressionof several Hoxb geneshas also been found in well as neuronsof the VIIIth cranial ganglion (Knowlton, 1967; the developing branchial archesand cranial ganglia in mouse. D’Amico and Noden, 1983).This would suggestthat the SOHo- Hoxb-2 and Hoxb-I are both expressedin branchial arch 2 and expressionseen later in the VIIIth cranial ganglion, by stage23, in the VIIIth cranial ganglion (Frohman et al., 1990; Hunt et The Journal of Neuroscience, February 1994, 14(2) 497 al., 199 l), which roughly correlates with their anterior bound- Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation aries of expression. It has been suggested that neural crest cells by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal already possess positional information prior to their migration Biochem 162:156-159. Cohen SM, Jtirgens G (1990) Mediation of Drosophila head devel- to form cranial ganglia and branchial arches in the form of a opment by gap-like segmentation genes. Nature 346:482485. combinatorial code of HOX genes (Hunt and Krumlauf, 199 1). Conlan RA, Rossant J (1992) Exogenous retinoic acid rapidly induces Classical transplantation experiments support this view; trans- anterior ectopic expression of murine Hex-2 genes in vivo. Devel- plants of presumptive branchial arch 1 neural crest cells into opment 116:357-368. Constantine-Paton M, Blum AS, Mendez-Otero R, Bamstable CJ (1986) the region of presumptive branchial arch 2 neural crest result A cell surface molecule distributed in a dorsoventral gradient in the in duplication of branchial arch 1 structures (Noden, 1983). perinatal rat retina. Nature 324:459462. Although SOHo- was expressedin the ,we find Crossland W, Cowan W, Rogers L, Kelly J (1974) The specification no evidence that the neural crest cells from this region express of the retino-tectal projection in the chick. J Comp Neural 155: 127- 164. SOHo- during migration to form the cranial ganglia and the D’Amico-Martel A, Noden DM (1983) Contributions of placodal and branchial arch 2. Since SOHo- is the first member of this class neural crest cells to avian cranial peripheral ganglia. Am J Anat 166: of homeoboxgenes to be found in vertebrates, the combinatorial 445468. models proposed for Hox cluster genesmay not apply to its de Long GR, Coulombre AJ (1965) Development of the retinotectal expressionpattern. In fact, SOHo- 2 appearsto be downstream topographic projection in the chick embryo. Exp Neurol 13:35 l-363. DiNardo S, Sher E, Heemskerk-Jongens J, Kassis JA, O’Farrell PH of the Hoxh genesin its temporal pattern of expression.Unlike (1988) Two-tiered regulation of spatially patterned engruiled gene Hoxb-I that is expressedin the migrating neural crest, which expression during Drosophila embryogenesis. Nature 332:604-609. will form the secondbranchial arch, SOHo- is expressed2 d Doe CQ, Hiromi Y, Gehring WJ, Goodman CS (1987) Expression after the neural crest cells arrive at the second branchial arch. and function of the segmentation gene fishi tarazu during Drosophila neurogenesis. Science 239: 170-l 75. The initial expressionof SOHo- I in the extreme ventral portion Doe CQ, Smouse D, Goodman CS (1988) Control of neuronal fate of the secondbranchial arch at stage23 followed by an expansion by the Drosophila segmentation gene evenskipped. 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