HOM/HOX genes are involved in the establishment of theanimal body plan by specifying the positional identity ofdifferent structures along the anteroposterior axis (reviewed inMcGinnis and Krumlauf, 1992; Krumlauf, 1994). It is alsobelieved that they contribute to the morphological diversitythroughout the animal kingdom. During evolution, Hox geneshave conserved both their organization in clusters and theordered correlation between the position of the genes in thecluster and their expression pattern along the body axis (spatialcolinearity) (Lewis, 1978; Graham et al., 1989; Duboule andDollè, 1989). From an evolutionary perspective, thedevelopment of a more complex body organization seems tocorrelate with the formation and amplification of Hox clusters,probably achieved through successive tandem duplications ofan ancestral homeobox-containing gene and subsequent clusterduplication (Field et al., 1988; Schugart et al., 1988; Kappenet al., 1989).
In agreement with this hypothesis, a single cluster with avariable number of Hox genes has been found in mostmetazoans, such as the Arthropods and Nematodes (reviewed
by Holland, 1992; Burglin and Ruvkun, 1993). In chordates,only one cluster is present in the genome of theCephalochordate Amphioxus (Holland et al., 1994) and it hasbeen suggested that the Urochordate Ciona intestinalis also hasa single cluster (Di Gregorio et al., 1995). Four apparentlyidentical Hox clusters are present in all mammals so faranalyzed (Boncinelli et al., 1988; McGinnis and Krumlauf,1992; Duboule, 1994); however, variation in the number andorganization of Hox clusters is observed in some lowervertebrates (Aparicio et al., 1997; Amores et al., 1998).
Genetic analysis of vertebrate Hox genes, involving bothloss- and gain-of-function mutations, has been carried outprimarily in the mouse through the use of gene targetingtechnology. Together, this large body of work has revealedimportant functional roles for the overlapping and orderedexpression patterns of Hox genes in many different tissues ofthe embryo, such as the CNS, axial skeleton, limbs and gut(reviewed by McGinnis and Krumlauf, 1992; Maconochie etal., 1996; Duboule, 1993). There are distinct individual rolesfor most Hox genes, but there is also evidence for synergisticinteractions and functional redundancy between members,which are most likely due to overlapping expression patterns
Hox genes play a fundamental role in the establishment ofchordate body plan, especially in the anteroposteriorpatterning of the nervous system. Particularly interestingare the anterior groups of Hox genes (Hox1-Hox4) sincetheir expression is coupled to the control of regionalidentity in the anterior regions of the nervous system,where the highest structural diversity is observed.Ascidians, among chordates, are considered a good modelto investigate evolution of Hox gene, organisation,regulation and function. We report here the cloning and theexpression pattern of CiHox3, a Ciona intestinalis anteriorHox gene homologous to the paralogy group 3 genes. In situhybridization at the larva stage revealed that CiHox3expression was restricted to the visceral ganglion of thecentral nervous system. The presence of a sharp posterior
boundary and the absence of transcript in mesodermaltissues are distinctive features of CiHox3 expression whencompared to the paralogy group 3 in other chordates. Wehave investigated the regulatory elements underlyingCiHox3 neural-specific expression and, using transgenicanalysis, we were able to isolate an 80 bp enhancerresponsible of CiHox3 activation in the central nervoussystem (CNS). A comparative study between mouse andCiona Hox3 promoters demonstrated that divergentmechanisms are involved in the regulation of these genes invertebrates and ascidians.
Patterning the ascidian nervous system: structure, expression and transgenic
analysis of the CiHox3 gene
Annamaria Locascio1,*, Francesco Aniello1,*, Alessandro Amoroso1, Miguel Manzanares2, Robb Krumlauf2
and Margherita Branno1,‡
1Department of Biochemistry and Molecular Biology, Stazione Zoologica ‘Anton Dohrn’, Villa Comunale, 80121 Naples, Italy2Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA,UK*These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])
Accepted 5 August; published on WWW 6 October 1999
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and structural conservation between paralogous genes. Hence,Hox genes have multiple roles in patterning diverse axialstructures.
Organisms belonging to the Chordate phylum share somecharacteristics, such as the presence of an axial notochordflanked by muscles, a dorsal hollow nervous system and aventral endodermal strand. Given the key role of Hox genes inregulation of the body plan, study of their expression patternsand regulatory regions in different Chordates may aid in theidentification of both homologous structures and novelstructures, thus shedding light on the evolution ofmorphological diversity. Therefore it is important tounderstand the regulation of restricted Hox expression indifferent species, as one suggested means of coupling this genefamily to a range of common or unique morphogenic processescould be through the conservation, modification and/oracquisition of short regulatory elements (Gellon and McGinnis,1998).
The expression and regulation of the Hox genes has beenstudied in detail in the CNS (reviewed in Keynes andKrumlauf, 1994; Rubenstein and Puelles, 1994; Maconochie etal., 1996; Lumsden and Krumlauf, 1996). In the vertebrateCNS, Hox genes belonging to paralogy groups 1 to 4 areexpressed with partially overlapping domains whose anteriorboundaries correlate with the subdivision of the hindbrain intosegmental units or rhombomeres (Hunt et al., 1991; Keynesand Krumlauf, 1994). Mutational analysis in the mouse hasshown that these rhombomere-restricted domains of Hoxexpression underlie functional roles in multiple steps ofsegmental patterning, as illustrated extensively for the group 1genes (Carpenter et al., 1993; Mark et al., 1993; Studer et al.,1996, 1998). The remaining cluster members (paralogygroups 5-13) are expressed along the entire spinal cord inprogressively more posterior domains that are also functionallyimportant (Tiret et al., 1998).
The use of a simpler model system than the highervertebrates might contribute to the study of Hox genes and oftheir role in the evolution of chordates. Ascidians have a basicbody plan and are considered prototypical chordates. Despitetheir simple organization, recent evidences have pointed outa certain degree of conservation in the genetic pathwaysinvolved in the specification of different body structuresbetween ascidians and vertebrates (Di Gregorio and Levine,1998).
During ascidian embryogenesis, the nervous systemdevelops by the formation of a neural plate, which invaginatesand seals dorsally leading to a hollow nerve cord in a processreminiscent of vertebrate neurulation. At the larval stage, theascidian nervous system comprises an anterior vesicle in thetrunk, containing the pigmented sensory organs and thus calledsensory vesicle, followed, more posteriorly, by a visceralganglion and a caudal nerve cord. This latter structure is devoidof neuronal cell bodies and lies above the notochord (Nicol andMeinertzhagen, 1991).
Recent experiments have demonstrated the usefulness of C.intestinalis as a model system for studying gene regulation(Corbo et al., 1997a,b). In the present report, we have focusedour attention on a C. intestinalis Hox gene belonging to theanterior group, sharing the highest degree of homology to theclass 3 Hox genes of vertebrates and thus referred to as CiHox3.The analysis of CiHox3 spatial and temporal expression
patterns during C. intestinalis embryogenesis revealed that itsmRNA is confined to the larval CNS and in particular to a well-defined region of the visceral ganglion. In an effort tounderstand the mechanisms involved in the regulation ofCiHox3 expression during development, we have usedelectroporation (Corbo et al., 1997b) to introduce reporterconstructs, containing different fragments from the 5′-flankingregion of the CiHox3 gene fused to the lacZ cistron, intofertilized Ciona eggs. With this approach, we identified anenhancer element responsible for the specific expression of thereporter gene in the nervous system.
Furthermore, the CiHox3 promoter was tested in transgenicmouse embryos and mouse Hox3 regulatory elements wereelectroporated in Ciona in order to address whether regulatoryelements of Hox3 genes have been conserved during chordateevolution.
MATERIALS AND METHODS
AscidiansAdult C. intestinalis were collected in the Bay of Naples by the fishingservice of the Stazione Zoologica. Gametes were used for in vitrofertilization and embryos were raised in filtered sea water at 16-18°C.Samples at appropriate stages of development were collected and usedfor whole extract protein or RNA extraction, or fixed for whole-mountin situ hybridization.
Isolation of cDNA and genomic clonesA cDNA library made from embryos at larva stage (Gionti et al.,1998) was screened using as a probe a genomic fragment, namedCiHbox1, previously characterized by Di Gregorio et al. (1995) andcontaining the homeobox sequence of CiHox3. A positive clone insertwas sequenced on both strands by the dideoxynucleotide terminationprocedure (Sambrook et al., 1989).
A C. intestinalis cosmid library, constructed by Reference LibraryDatabase (RLDB, MPI for Molecular Genetic, Berlin-Dahlem,Germany; Burgtorf et al., 1998), was screened at high stringency usingas a probe the [32P]oligonucleotide 5′-TCTATCGGCAGCCATA-AGAGTC-3′, complementary to the most 5′ coding region of thecDNA sequence. Three positive clones: MPMGc119L0224,MPMGc119B058 and MPMGc119D1338 were amplified and DNAwas purified with QIAGEN kit (Qiagen Inc., Chatsworth CA, USA).The genomic inserts were subjected to digestion with the restrictionendonucleases EcoRI or XbaI followed by Southern blot hybridizationwith the oligonucleotide described above. An XbaI- and an EcoRI-positive fragments of about 4 kb and 1 kb in length, respectively,derived from the cosmidic clone MPMGc119B058 and containing the5′ coding region of CiHox3, have been subcloned in the pBlueScriptII KS vector and sequenced.
The 4.2 kb promoter region was obtained by assembling threepartially overlapping EcoRI or XbaI genomic fragments obtainedfrom the successive hybridization of Southern blot of the cosmidicclone MPMGc119B058 with oligonucleotides at the 5′ end of eachfragment.
Introns position and length have been determined by PCR using thesame cosmid clone as template and useful primers complementary tothe cDNA sequence.
RNA preparation and northern blottingTotal RNA from various stage embryos was prepared according toChomczynski and Sacchi (1987). Poly(A)+ RNA was purified byoligo(dT)-cellulose chromatography (Sambrook et al., 1989).Northern blot hybridization was carried out as described by Gionti etal. (1998) using 15 µg of poly(A)+ RNAs per lane and a 32P-labeled
A. Locascio and others
4739CiHox3: expression pattern and regulation
CiHox3 cDNA insert as a probe. In addition, a Ci-CaM cDNA (DiGregorio et al., 1998) probe was used as a control.
In situ hybridizationTwo Dig-11-UTP-labeled RNA probes were used for whole-mount insitu hybridization experiments: the CiHox3 cDNA insert and a DNAfragment, amplified by PCR, corresponding to the 5′ coding region ofCiHox3 (extending from position −70 to +500). Conditions for in vitrotranscription were as described by the manufacturers in theDigoxigenin RNA Labeling Kit (Boehringer-Mannheim). Whole-mount in situ hybridizations on Ciona embryos and larvae werecarried out as described in Caracciolo et al. (1997). Semithin sectionswere performed as described by Di Gregorio et al. (1998).
RNase protection analysisThe transcriptional initiation sites were determined by RNaseprotection assay basically according to the RNase protection kitinstructions (Boehringer Mannheim).
A 353 bp fragment (spanning −179 bp to +174 bp), amplified byPCR, was cloned in the EcoRV site of the pBluescript II KS vector.The plasmid was digested with XbaI and used as template for in vitrotranscription of [32P]UTP-labeled antisense riboprobe. 5 µg ofpoly(A)+ RNA from embryos at larva stage were hybridized overnightat 45°C with the probe. After RNase A and T1 digestion, the protectedfragments were run on 8% denaturing polyacrylamide gel along witha reference sequence for size determination.
Preparation of constructsThe basic electroporation vector was pBlueScript II KS containing thelacZ and SV40 polyadenylation sequences (pBS+LacZ). Genomicfragments of 0.2, 0.45, 1.0 and 1.5 kb were amplified by PCR fromthe cosmid clone MPMGc119B058. The amplified fragments wereligated with the pBS+LacZ vector in the 5′ to 3′ orientation. An XbaI-KpnI CiHox3 genomic fragment, obtained from the cosmid cloneMPMGc119B058, was subcloned in the 0.45 construct, XbaI andKpnI digested, to obtain the construct 3.0.
The 4.2 construct was obtained by cloning an XbaI (end-filled)-XbaI genomic fragment, from the same cosmid clone, in the KpnI(end-filled)-XbaI-digested 0.45 construct.
To generate the internal deletion mutants ∆4-3 and ∆3-25 theregions from −3 kb to −0.5 and from −2.5 kb to −0.5 kb of the 4.2and 3.0 constructs, respectively, were removed by KpnI and SnaBI oronly SnaBI digestions and the resulting linear DNA fragments wereself-ligated. The deletion mutant ∆3-2 was generated by exonucleaseIII digestion of the region from −2 kb to −0.45 kb of the 3.0 constructaccording to manufacturer’s instructions (Promega). All the otherconstructs, P-1 to P-6, were obtained by cloning the correspondinggenomic fragments, amplified by PCR, in the end-filled HindIII siteof the 0.45 construct.
For transgenic mice, a 2.3 kb KpnI-HindIII fragment (construct 1,Fig. 7A) or a 500 bp PCR product (construct P1, Fig. 7A) from theCiona Hox3 genomic region were cloned downstream of a lacZreporter expression construct (pBGZ40) containing the human β-globine promoter (Yee and Rigby, 1993). DNA purified from vectorsequences was microinjected in mouse oocytes, and lacZ expressionin embryos detected as described by Whiting et al. (1991).
The mouse Hoxb3 and Hoxa3 constructs used for electroporationin Ciona embryos were the constructs N. 5 and 6 by Manzanares etal. (1997) and #1.4, #3.3 (Manzanares et al., 1999).
ElectroporationElectroporation of fertilized and dechorionated C. intestinalis eggswas as described in Corbo et al. (1997b) with a few modifications:capacitance setting was between 700 and 850 µF so that the pulserange was 15-18 mseconds and the final volume in the 0.4 cm cuvetteswas 700 µl.
Embryos were allowed to develop at 16°C, in fresh sea water in
0.9% agarose-coated dishes, until the required stage, fixed in 1%glutaraldehyde in sea water for 30 minutes at room temperature,washed twice in PBS 1× and stained at 30°C in a solution containing3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1 mM MgCl2, 0.1% Tween20and 200 µg/ml X-gal in PBS.
Band-shift assayBand-shift assay was carried out essentially as described by Yuh et al.(1994) with 8 µg of protein extracts from embryos at larva stage, 50ng of poly(dIdC) and 4 fmol of 32P-labeled AL2 oligonucleotide. Thesequence of the random oligonucleotide was: 5′-CTGCTTTGA-TGGATGGAGCTG-3′. Protein extracts were prepared according toTomlinson et al. (1990).
RESULTS
Isolation CiHox3 geneCiHox3 was isolated from a cDNA library prepared withpoly(A)+ mRNA from C. intestinalis larvae using a genomicfragment, containing the homeobox sequence (previouslynamed CiHbox1 in Di Gregorio et al., 1995) as a probe.
Sequence analysis of the isolated cDNA clone, 1994 bp inlength, showed that the translation start codon was missing. Toobtain the remaining 5′ coding sequence, a cosmid DNAlibrary, prepared by the RLDB (Burgtorf et al., 1998), wasscreened with an oligonucleotide complementary to theanteriormost region of the cDNA. Three positive clones wereisolated and analyzed, and an EcoRI fragment from the cosmidclone MPMGc119B058, partially overlapping with the cDNA,was subcloned and sequenced. This fragment was found tocontain the remaining 5′ coding region, preceded by a seriesof stop codons, and part of the promoter sequence of CiHox3.The analysis of the genomic organization of CiHox3 has beenperformed by PCR amplification on the cosmid clones usingoligonucleotides complementary to the CiHox3-coding region.The complete genomic structure of CiHox3 thus obtained isschematically reported in Fig. 1A. It includes two introns of2700 and 2173 bp respectively, and three exons including the5′ (90-167 bp) and the 3′ UTR (280 bp) regions, and a codingregion of 2199 bp, encoding for a putative protein of 733 aminoacids.
It is interesting to note that the first intron, positionedbetween the sequences encoding the hexapeptide and thehomeodomain, is conserved among all Hox3 genes ofchordates, while the presence of an intron in the homeoboxsequence seems to be a peculiar feature of C. intestinalishomeobox-containing genes (Di Gregorio et al., 1995).
The comparison of the deduced amino acid sequence ofCiHox3 with those deposited in the GenBank and EMBLdatabases revealed the highest degree of homology with thehomeodomains of the paralogy group 3 HOX proteins. Acomparison of the homeodomain, the hexapeptide and theinterposed sequence of CiHox3, AmphiHox3, the HOX3proteins of mouse and the Drosophila Proboscipedia is shownin Fig. 1B. The highest percentage of identity was found in thehomeodomain of AmphiHox3 (81%) and the lowest in thehomeodomain of Proboscipedia (72%). Interestingly, thedegree of homology (78%) is the same among the three HOXgroup 3 proteins of mammals (HOXa3, HOXb3 and HOXd3).
A lower degree of conservation can be identified in theregion interposed between the homeodomain and the
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hexapeptide where 10-12 identical amino acids are conserved.In this domain, CiHox3 does not include the stretch of glycineresidues, characteristic only of mouse and human HOXb3,suggested to have a hinge function (Beachy et al., 1985; Shamet al., 1992). Therefore this may represent characteristicsequence acquired only by the HOXb3 proteins later inevolution.
To establish the site of transcript initiation, RNase protectionassay was carried out on poly(A)+ RNA from the larval-stageembryos using a [32P]UTP-labeled riboprobe which extendsfrom position −179 to +174 and includes both part of the 5′coding region and of the adjacent promoter sequence. Threealternative transcription start sites were found (Fig. 1C):two giving a stronger signal corresponding to position −90 and
A. Locascio and others
Fig. 1. Structure of the CiHox3 gene, amino acid comparison of the homeodomain and transcription initiation sites. (A) The coding sequenceof 2199 bp in length is boxed and includes the homeobox (white box). Interrupted lines indicate the two introns of about 2700 and 2173 bpin length. 5′ and 3′ UTR are shown in striped boxes. The 5′ genomic region is presented in bold line and is reproduced in magnified view toshow the alternative transcription start site, indicated by arrows, and the putative TATA and CAAT boxes (black and white dots, respectively).The bar −179 +174 indicates the 353 bp fragment used for the RNase protection assay. (B) The sequences shown in the comparison are asfollows: CiHox3 (C.i., Ciona intestinalis; accession no. AJ132778), AmphiHox3 (B.f., Branchiostoma floridae; accession no. A49127),Hoxd3, Hoxb3 and Hoxa3 (M.m., Mus musculus; accession no. U03485; P09026; Q61197) and Proboscipedia (D.m., Drosophilamelanogaster; accession no. P31264). The identical residues are indicated by a dash. To optimize continuity of conserved regions gaps,indicated by dots, are introduced. Number of glycine residues in the interposed region between the hexapeptide and the homeodomaincharacteristic of mouse HOXb3 are indicated by numbers in brackets. Per cent of identity to the ascidian homeodomain are given in the rightcolumn. (C) CiHox3 mRNA start sites were determined using poly(A)+ RNA from embryos at larva stage. Lanes A, C, G, T are sequencingreactions of unrelated DNA used for size determination. Lane 1, undigested probe corresponding to the 353 bp antisense probe; lane 2,protected fragments obtained after RNase T1 and RNase A digestion are indicated by arrows and correspond to alternative transcription startsites 167, 101 and 90 bp upstream from the translation start codon.
4741CiHox3: expression pattern and regulation
−101, and a third one giving a weaker signal at position −167from the translation start codon (Fig. 1A). The same result wasobtained using different riboprobes or poly(A)+ RNAs fromtailbud-stage embryos (data not shown).
CiHox3 expression in the CNS of the Ciona embryoThe timing of CiHox3 expression during C. intestinalisdevelopment was determined by northern blot analysis. Usingthe cDNA clone as a probe, no signal was detected in stagesprior to the early tailbud in which a single positive band ofabout 2.6 kb appears (Fig. 2A). The signal remained faint inthe middle tailbud and gradually increased in the followingstages of development, peaking in the swimming larva. Thesame northern, hybridized with the Ci-CaM cDNA (DiGregorio et al., 1998), served as a control for RNA loading(Fig. 2B).
In situ hybridization assays were then carried out todetermine the localization of CiHox3 message during Cionaembryogenesis. No signal could be detected even after longstaining times in early tailbud embryos probably due to the lowabundance of the message (not shown). CiHox3 expressionbecame visible at larval stage and was restricted to the nervoussystem (Fig. 3A,B). In particular, the transcript was localizedin the anteriormost region of the visceral ganglion showingwell-defined both anterior and posterior limits. Transversesections, cut at different levels of the hybridized larva,confirmed this conclusion. In particular, staining seemed to beconfined in the lateral and most superficial region of thevisceral ganglion (Fig. 3C) which, according to the studies byNicol and Meinertzhagen (1991), contains the cellular bodiesof ganglion cells. No signal was detected outside the CNS.
CiHox3 promoter analysis in electroporatedembryosAs indicated in Fig. 1A, the CiHox3 promoter region containsseveral putative TATA and CAAT boxes in the region adjacentto the three alternative transcription start sites. In order toidentify the elements required for neural-specific expression ofCiHox3, the 5′ genomic sequence of this gene was furtherexamined using an electroporation method (Corbo et al.,1997b). Initially, we assayed the 4.2 kb genomic DNA
fragment starting immediately upstream from CiHox3translation start site and linked to a lacZ reporter gene (Fig.4A). The electroporated Ciona embryos were allowed todevelop until the stage of interest, they were then fixed andassayed for β-galactosidase activity by X-gal staining. Asshown in Fig. 4C, this construct drove the expression of thereporter gene in the nervous system of the larvae. In particular,staining was visible in the visceral ganglion showing virtuallythe same localization as the endogenous transcript. However,this construct also showed ectopic expression in theanteriormost region of the nervous system, at the level of thesensory vesicle and around the otolith. In about 5% of theelectroporated embryos, the trunk mesenchyme was alsoectopically stained (not shown). The onset of expression wasat early tailbud stage (Fig. 4B). In these embryos, stainingbecame visible in the anterior region of the developing nervoussystem after 10 days of staining. Three internal deletiontransgenes and a series of deletion constructs progressivelytruncated at the 5′ end were then prepared and their effects wasanalyzed (Fig. 4A). The same pattern of expression found withthe 4.2 kb transgene was obtained only with the construct 3.0,while the progressively shorter constructs of 1.5, 1.0, 0.45 andthe three deletion mutants ∆4-3, ∆3-25 and ∆3-2 were unableto give expression in the nervous system. These constructs,however, showed strong ectopic labeling in the trunkmesenchyme at the larval stage (Fig. 4E). The embryoselectroporated with the 0.45 construct were also analyzed atearlier stages of development and after 2 weeks of stainingmesenchymal cells of the neurula stage embryos were weaklylabeled (not shown). During the subsequent stages ofdevelopment, the signal became stronger and was clearlyrecognizable after one week of staining in the mesenchymalpockets of the early tailbud stage embryo (Fig. 4D). Thesmallest deletion construct 0.2 (shown in Fig. 4A), containingonly the putative TATA and CAAT boxes was unable to drivethe expression of the reporter gene (data not shown).
Identification of a neural-specific CiHox3 enhancerand comparative study of Ciona and mouse Hox3promotersThe analysis of the results obtained with the constructs shownin Fig. 4A suggested that the region from −2 kb to −1.5 kb(indicated as red box) of the promoter contained elementsresponsible for the restricted expression of the lacZ transgenein the larval nervous system. To confirm this hypothesis aconstruct (P1, shown in Fig. 5A) was prepared by fusing thisregion upstream from the 0.45 construct of Fig. 4A. The β-galactosidase activity in the embryos electroporated with thisconstruct was assayed at the larval stage. The reporter gene wasexpressed both in the visceral ganglion and the sensory vesicleof the CNS (Fig. 5C) thus reproducing the result obtained withthe 4.2 and 3.0 constructs of Fig. 4C. Also in this case about5% of the embryos showed ectopic staining in the trunkmesenchyme (not shown). These results indicate that thesequence contains all the element(s) necessary to activatetranscription in the CNS.
In an attempt to isolate the minimal sequence responsible forthe neural-specific expression pattern, the P1 sequence wassubdivided in a series of smaller and partially overlappingfragments obtained by PCR, which were subcloned upstreamfrom the 0.45 construct as shown in Fig. 5A.
Fig. 2. Northern blot analysis of CiHox3. Each lane contains 15 µgof poly(A)+ RNA from Ciona eggs and embryos at the indicatedstages of development. Blot was hybridized with a 32P-labeledCiHox3 cDNA (A) and with the Ci-CaM cDNA probe (B) ascontrol.
4742 A. Locascio and others
Fig. 3. In situ localization of CiHox3 transcript at the larva stage. Whole-mount Ciona larvae were hybridized with digoxigenin-labeledCiHox3 antisense RNA probe and stained with alkaline phosphatase. (A,B) Lateral and dorsal view of a larva showing CiHox3 expression inthe visceral ganglion (arrow). Line s indicates level of transverse section. (C) Transverse section at the (s) level of the visceral ganglion of ahybridized larva. CiHox3 mRNA expression is observed in the most external lateral cells of the ganglion. cg, ganglion cells; En, endoderm;oc, ocellus; ot, otolith; sv, sensory vesicle; vg, visceral ganglion.
Fig. 4. Summary of CiHox3transgene constructs and theirexpression in electroporatedCiona embryos. (A) Diagramof different 5′ CiHox3promoter sequences analyzedin the electroporatedembryos. At the top, genomicsequence of CiHox3 genewith the coding regionrepresented as a widerectangle. The restrictionsites (H, HindIII; E, EcoRI;K, KpnI; S, SnaBI; X, XbaI)used for the isolation of thepromoter region and theconstruction of thetransgenes are also indicated.BamHI (B) was introduced
by PCR. Right side, construct names and tissueswhere the reporter gene is expressed. The red boxindicates the minimal element necessary for theexpression in the Ciona CNS. All fragments startimmediately upstream from the translation start codonof CiHox3 and extend in the 5′ direction for theindicated length. Constructs ∆4-3, ∆3-25 and ∆3-2 areinternal deletion mutants where the dot line representsthe deleted region. (B) Embryo at tailbud stageelectroporated with construct 4.2. X-gal staining isvisible after 12 days in the anterior region of thedeveloping nervous system. (C) Larva electroporatedas in B, where the signal is present, after 8-9 days, inthe visceral ganglion and ectopically in the sensoryvesicle. (D,E) Embryos at tailbud and larva stageselectroporated with 0.45 construct. The staining is lostfrom the nervous system and is found, after 3-6 days,in ectopic localization in the mesenchyme. ans,anterior nervous system; mp, mesenchymal pocket;tm, trunk mesenchyme; sv, sensory vesicle; vg,visceral ganglion.
4743CiHox3: expression pattern and regulation
Embryos electroporated with the construct P2 presentedonly ectopic staining in the trunk mesenchyme of the larva.Constructs P3 and P4 were able to drive expression of lacZ inthe larval nervous system, both in the visceral ganglion andectopically in the sensory vesicle (not shown). The P4sequence was further subdivided into two fragments andembryos electroporated with the construct corresponding to P5did not show expression in the nervous system while P6fragment reproduced the result obtained with P4 (Fig. 5D).Therefore, the element(s) necessary for the expression in theCNS are present in the 80 bp of the 5′ flanking region fromposition −1943 to −1864 (Fig. 6A).
In order to study the ability of the 80 bp sequence to bindnuclear proteins, different overlapping oligonucleotides of 20-24 bp each were used in gel shift assays on whole-cell extractsof Ciona larvae. Only the oligonucleotide AL2, indicated inFig. 6A, was able to form a specific complex (Fig. 6B). In fact,
a 200-fold excess of cold AL2 was able to decrease complexformation while an unrelated oligonucleotide had no effect.Two oligonucleotides, partially overlapping with AL2, namedAL1 and AL3 (Fig. 6A), were tested for the ability to competewith AL2 for complex formation. As shown in Fig. 6B, onlyAL1 was able to decrease the binding, suggesting that thecommon region between the AL1 and AL2 oligonucleotideswas involved in the binding. The analysis of the AL2 sequencewith the MATINSPECTOR 2.2 and TRANSFAC databases didnot reveal any likely recognition sequence for knowntranscription factors.
To date, only the CNS-specific regulatory elements of mouseand chicken Hoxb3 or mouse Hoxa3 (Manzanares et al., 1997,1999) promoters have been studied and several kreisler-likeelements have been found to be involved in their activation inrhombomeres (r) 5 and 6. Therefore, we decided to test thetranscriptional activity of CiHox3 regulatory elements
Fig. 5. Analysis of the 500bp genomic sequencecontaining the CiHox3enhancer element(s) and ofthe mouse kreisler bindingelements. (A) On the left,diagram of the 500 bp regionand of derived fragmentsstudied in the electroporationexperiments. The red boxindicates the 80 bpcontaining the minimalelement necessary for theCNS expression. Constructsnames and their expressionterritories are shown on theright. (B) On the left,diagram of the mousekreisler binding element forthe HOXa3 and HOXb3promoters. The green boxesindicate the localization ofthe kreisler elements.Constructs names and theirexpression territories areshown on the right. Larvaeelectroporated with the P1construct (C) and with the P6construct (D). X-gal stainingis visible in the visceralganglion and in the sensoryvesicle. sv, sensory vesicle;vg, visceral ganglion.
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in mouse embryos. Forthis purpose, reporterconstructs with lacZ formicroinjection in mouseoocytes were prepared.We first tested a 2.3 KpnI-HindIII fragment (Fig. 7A), which includes the regioninvolved in the neural-specific expression of CiHox3in Ciona embryos. Transgenic embryos for thisconstruct consistently gave reporter expression in apattern reminiscent of mouse Hox regulatoryelements. Expression was detected in the ventralportions of rhombomere (r) 4 and also in a domainwith an anterior boundary at the r6/r7 boundary thatextended posterior into the spinal cord (Fig. 7B,C). Wethen tested in mouse embryos the CiHox3 P1 promoterfragment that was responsible for specific lacZexpression in the Ciona nervous system (Fig. 7A). Incontrast to the first case, this construct did not generateany specific pattern of expression in transgenic mouseembryos, but showed only ectopic expressionpresumably due to effects of the integration site (datanot shown). Therefore, these results suggest that theCNS-specific regulatory elements from CiHox3 genethat direct reporter expression in that species arenot the ones recognized by the mouse embryotranscriptional machinery. Hence, other elements mustbe present in Ciona genomic fragments that are ableto mediate a segmental expression in the mousehindbrain. These elements might correspond to auto- or cross-regulatory regions that are capable of responding toendogenous mouse Hox genes (see Discussion).
Conversely, when the mouse kreisler-dependent enhancerregions, involved in the expression of Hoxa3 and Hoxb3 in thehindbrain (schematized in Fig. 5B), were electroporated inCiona embryos, no nervous-system-specific expression wasobserved. Only ectopic expression was detected, such asstaining in mesenchymal cell using Hoxa3 and in the muscleof the tail using Hoxb3 (data not shown). Hence the mousekreisler-like response elements, conserved in highervertebrates, seem unable to regulate any part of a CiHox3-typeexpression pattern in Ciona. It is possible that other CNSregulatory regions recently identified in the mouse Hoxa3 locus(Manzanares et al., 1999) might function in Ciona. However,our results reveal that the regulation of Hox3 gene expressionin the mouse and Ciona CNS does not appear to be mediatedby the same highly conserved elements, as either of theenhancers shown to direct specific expression in one speciesfails to direct reporter expression in the other. This mightindicate that different regulatory elements and components areutilized by these species and/or that similar components areinvolved but they have slightly diverged and are unable tofunction across such an evolutionary distance.
DISCUSSION
We have isolated a homeobox-containing gene called CiHox3encoding for a protein with 81 and 78% of identity in itshomeodomain and hexapeptide sequences with AmphiHox3and HOX paralogy group 3 of vertebrates, respectively (Fig.
1B). The temporal and spatial expression pattern of CiHox3were analyzed by northern blot and whole-mount in situhybridization. The earlier stage at which a clear signal couldbe detected in the northern blot was the early tailbud. CiHox3mRNA localization was restricted to the larval nervous systemand, in particular, to the visceral ganglion.
The larval nervous system in C. intestinalis can besubdivided into three regions, which have been attributeddistinct functions (Nicol and Meinertzhagen, 1991): in thetrunk, an anterior sensory vesicle, containing the two sensoryorgans, followed by the visceral ganglion and, in the tail, thespinal cord. The visceral ganglion has been considered theintegrating center of the ascidian nervous system and itsarchitecture has been studied in detail and was found tocontain, in its outer region, neuronal cell bodies (ganglioncells), while in the inner part are present fibrous neuropil(Barnes, 1971). Transverse sections of hybridized larvae (Fig.3C) showed that CiHox3 message was localized in the externalarea of the visceral ganglion suggesting it could be expressedin the neuronal cell bodies. The comparison of Hox3expression territories between ascidians, cephalochordates andvertebrates might shed light on the evolution of these genesalong this phylogenetic line and could permit the establishmentof structural homologies among such different taxa. Studies onAmphioxus AmphiHox3 expression revealed that it is restrictedto a specific domain of the dorsal nerve cord with a sharpboundary only in the anterior part, in particular, at the level ofsomite pairs 4 and 5 and it is also expressed in the posteriormesoderm (Holland et al., 1992). In vertebrates, thelocalization of paralogy group 3 genes transcripts is notconfined to the nervous system, where they are expressed inthe hindbrain with anterior limit at the level of rhombomere 5,
A. Locascio and others
Fig. 6. Gel-shift assay withAL2 double-strandedoligonucleotide and proteinextracts from Ciona larvae. (A) Nucleotide sequence of theregulatory region contained inthe P6 construct. Theunderlined sequences AL1,AL2 and AL3 correspond tothe oligonucleotides used inbandshift assays. (B) A single-shifted band is observed inlane 2 where no coldcompetitor was added.Incubation with 200-fold molarexcess of oligonucleotides AL2or AL1, but not AL3 orrandom oligonucleotide (R),substantially reduced thecomplex formation.
4745CiHox3: expression pattern and regulation
but are also detectable in posterior and lateral mesoderm (Huntet al., 1991). Therefore, our analysis on CiHox3 expression,which is restricted to the nervous system, demonstrates that thecommon feature of Chordates Hox3 genes is in the spatialpatterning of the nervous system.
Fig. 8 shows a schematic representation of the territories ofexpression of CiHox3 and Cihox5, the Ciona Hox genes so faranalyzed in the larva, compared with the neural expression ofmouse Hox counterparts. A previous study on the Cionahomologue of the paralogy group 5 genes, Cihox5 (Gionti atal., 1998), revealed that its expression is limited to the larvaneural tube.
This demonstrated that the spatial colinearity rule, i.e. theexpression pattern of Hox genes follows their position in thecluster, is fulfilled in Ciona. As shown in the scheme (Fig. 8),CiHox5 is expressed in the most anterior region of the larvalneural tube, therefore posteriorly to CiHox3 and withoutoverlapping domains.
Moreover, their territory of expression has well-definedanterior and posterior limits, feature which, together with theirrestricted expression to the nervous system, seems to be uniqueto Ciona Hox genes. In cephalochordates and vertebrates, Hoxgenes are present in different mesodermal regions and theirneural expression has only a defined anterior limit, whileposteriorly extends and overlaps along the entire spinal cord.
Previous studies suggested that ascidians neuraldorsoventral patterning (Corbo et al., 1997a; Glardon et al.,
1997; Wada et al., 1997) and neural anteroposterior patterningare regulated by the same mechanisms described in vertebrateCNS. Wada et al. (1998) have proposed a subdivision of theascidian nervous system based on the comparison of theterritories of expression of genes involved in the patterning ofanteroposterior structures of vertebrates and their ascidiancounterparts.
Our results indicated that CiHox3 is expressed in Ciona
Fig. 7. Analysis of CiHox3 promoterregions in transgenic mouseembryos. (A) Diagram of CiHox3promoter fragments fused to thereporter lacZ gene and summary oftheir territories of expression intransgenic mice. N, indicates thenumber of mouse embryos analyzed.(B,C) Lateral and dorsal view of amouse embryo microinjected withconstruct 1. ov, otic vesicle; sc,spinal cord.
Fig. 8. Comparison of Hox3 and Hox5 expression in the nervoussystem of a C. intestinalis larva and a mouse embryo. Hox3territories of expression are shaded, Hox5 are striped. Theexpression domain of Hox3 genes in the CNS of a murine embryo(right side) has an anterior boundary in the hindbrain at the r4/r5border and posteriorly extends along the spinal cord. Thehomologous CiHox3 gene is expressed, in the anterior region of thevisceral ganglion of the Ciona larva (left side) with well-definedanterior and posterior limits. Murine Hox5 genes are expressedexclusively in the spinal cord while CiHox5 mRNA was found inthe neural tube of the larva. Note the presence of a posterior limitonly for the Ciona Hox5 gene. The expression domains of murineHox3 and Hox5 genes in tissues other than the neural tube is notshown. The murine embryo was from Lumsden (1990). nt,notochord; r, rhombomeres; sc, spinal cord; sv, sensory vescicle; vg,visceral ganglion.
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visceral ganglion and are in agreement with the suggestion thatthe mechanisms involved in the anteroposterior regionalizationof the nervous system have been conserved between ascidiansand vertebrates.
In Halocynthia roretzi, the expression pattern of HrHox1was analyzed by Katsuyama et al. (1995). In the nervoussystem of this ascidian, which lacks a defined visceralganglion, its expression is limited in the larva to the sensoryvesicle and to the junction of trunk and tail. It has beensuggested that this latter localization corresponds to the regionof the visceral ganglion of other ascidian species (Wada et al.,1998). Since HrHox1 territory of expression is equivalent tothe visceral ganglion, there would be an overlapping ofexpression with CiHox3. Therefore, the study of C. intestinalisHox1 and Hox2 homologues would be necessary to comparethe expression territories of Ciona and Halocynthia Hox genes.
To better understand the evolutionary relationships of Hox3genes between ascidians and vertebrates, we carried out theanalysis of CiHox3 promoter regulatory elements with theelectroporation method. The results indicated that a sequenceof 80 bp, from position −1943 to −1864 from the translationstart site, contained the positive regulatory element(s) able toactivate transcription in both the visceral ganglion and thesensory vesicle.
Analysis of this sequence by band-shift assay indicated thatonly the AL2 sequence (Fig. 6A) was able to form a complexwith protein extracts from Ciona larvae. This suggested thepresence of an unknown transcription factor binding site in theAL2 sequence presumably involved in the neural-specificactivation of CiHox3.
It has been proposed that the conservation of a clusterorganization for Hox genes during evolution was also due tothe presence of common regulatory elements for different Hoxgenes (Lufkin, 1996). Therefore, we decided to see whether theregulatory elements identified from CiHox3 would berecognized by vertebrate transcriptional networks and, on thecontrary, whether the mouse HOXa3 and HOXb3 promoterswere able to work in Ciona. In fact, although there is ampleproof of the common evolutionary origin of Hox complexesthroughout all animal phyla, this has been studied basically onthe coding regions for the Hox genes. Much less is knownabout the conservation and/or divergence of regulatoryelements. The regulation of vertebrate group 3 Hox genes hasrecently been analyzed, and it has been shown that the b-ZIPtranscription factor encoded by the kreisler gene is responsiblefor the upregulation of Hoxb3 in r5 and Hoxa3 in r5 and r6(Manzanares et al., 1997, 1999). Therefore, direct comparisonsbetween regulation of mouse and Ciona group 3 Hox genes ispossible.
Mouse Hoxa3 and Hoxb3 rhombomere-specific regulatoryelements tested in Ciona embryos were unable to reproduceany neural-specific expression. This suggests that indeed theregulation of Hox3 genes between mouse and Ciona issomewhat different. However, the kreisler-dependentenhancers could represent a recent acquisition by the highervertebrates, which occurred during the early duplicationsleading to four Hox complexes. It is possible that other CNScontrol regions from the mouse group 3 loci, for example fromHoxd3 or those recently isolated in Hoxa3 (Manzanares et al.,1999), might be able to function in Ciona. Since Ciona isbelieved to have only one complex, its group 3 CNS elements
might have been differentially distributed amongst the multiplemouse group 3 members.
The converse experiment, testing two different CiHox3promoter fragments in transgenic mouse embryos for theirability to drive reporter lacZ expression, showed that they didnot function effectively in mice. The smallest fragment thatdrives nervous system expression in Ciona was negative inmice. This again suggests a divergence of control elements ormechanisms between these species. While this may arise dueto totally different mechanisms, it is also possible that smalldivergence of the binding site sequences, and/or associatedchanges in the binding capabilities of the same trans-actingfactors, would render Ciona sites unrecognizable by the mousemachinery or vice versa.
Nevertheless, it is interesting that when a slightly largerCiona enhancer fragment was tested, a reproducible segmentalpattern of expression in the mouse hindbrain was obtained.This consisted of expression in the ventral portions of r4 anda second domain starting at the r6/r7 boundary, which spreadsposteriorly into the spinal cord. This pattern is reminiscent ofthose seen using the mouse Hoxb1 r4 enhancer (Marshall et al.,1992; Popperl et al., 1995), and the Hoxb4 r6/7 region Aenhancer (Whiting et al., 1991). One possible explanation forthe similarities in these patterns, comes from the observationthat both the Hoxb1 r4 and the Hoxb4 r6/7 domains, aredependant on auto-/cross-regulatory loops involving Hox/Pbxinteractions (Popperl et al., 1995; Gould et al., 1997).Therefore, the larger CiHox3 CNS enhancer that also functionsin mice, might contain both the minimal CNS element definedin this study and an auto-/cross-regulatory element. The patternobtained in mice would then reflect a read-out of mouseHox/Pbx complexes acting through a CiHox3 Hox-responsiveelement. This may indicate that the Ciona Hox complex alsouses auto-/cross-regulatory interactions for maintaining laterexpression patterns. In conclusion, this comparative regulatoryanalysis between Ciona and mice demonstrates that, at leastfor the group 3 Hox genes, there is considerable divergence insequences implicated in mediating CNS expression. Furtheranalysis in other chordates and intermediate species will berequired to elucidate the molecular basis of these differences.
We are indebted to Dr Albertina Fanelli for her invaluable help inthe preparation of the manuscript. We thank Professor Roberto DiLauro and Dr Rosaria De Santis for helpful discussion and GennaroIamunno for sectioning C. intestinalis larvae. We thank AmandaHewett, Peter Mealyer and Rosemary Murphy for help in animalhusbandry. M. M. was supported by HFSP and EEC Marie Curiepostdoctoral fellowships and by an EEC Biotechnology Networkgrant (#BIO4 CT-960378). This work was also funded in part by CoreMRC Programme support and an EEC Biotechnology Network grant(#BIO4 CT-960378) to R. K.
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