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Isolation of DNTNP, Which Encodes a Potential NuclearProtein That Is Expressed in the Developing, DorsalNeural TubeLIN JUN,1 ARMAND L. BALBONI,1 JEFFREY T. LAITMAN,2 AND ANDREW D. BERGEMANN1*1The Lillian and Henry M. Stratton-Hans Popper, Department of Pathology, Mount Sinai School of Medicine,New York, New York2Center for Anatomy and Functional Morphology, Mount Sinai School of Medicine, New York, New York

ABSTRACT We have performed a screen toidentify genes expressed in a functionally signif-icant anatomic region of the vertebrate dorsalneural tube, the dorsomedial roof of the thirdventricle (DMRTV). The DMRTV includes the pri-mordia of a series of circumventricular organs.The screen searched for genes preferentially ex-pressed in the DMRTV of stage 18–25 chickenembryos, relative to their telencephala and ven-tral diencephalon. Through this screen, we havecloned a series of genes strongly expressed in thedorsal but not ventral neural tube. We describehere the first of these genes, DNTNP (dorsal neu-ral tube nuclear protein). DNTNP is highly ex-pressed in the dorsal regions of the diencepha-lon, the midbrain, the hindbrain, and the spinalneural tube in the chicken stage 18 embryo. Ex-pression is also observed in the telencephalon,the branchial arches, the heart, and somites, butis absent from the presomitic mesoderm. Theamino acid sequence of DNTNP reveals that itbelongs to an uncharacterized protein familywith at least two additional members. All themembers of this family possess a basic regionreminiscent of a nuclear localization signal(NLS). We demonstrate that the putative NLS ofDNTNP can indeed direct nuclear localization ofgreen fluorescent protein (GFP). The dorsal lo-calization of DNTNP in the early embryonic cen-tral nervous system suggests roles for this mole-cule in specifying dorsal cell fates within theneural tube. © 2002 Wiley-Liss, Inc.

Key words: circumventricular organ; neuraltube; neuroendocrinology; nuclearlocalization

INTRODUCTION

The cell fate of precursor cells within the vertebratecentral nervous system is in large part determined bythe intersection of a dorsal-ventral patterning systemwith a rostral-caudal patterning system (Lumsden and

Krumlauf, 1996; Jessell, 2001). The dorsal-ventral sys-tem is derived from an antagonistic relationship be-tween ventralizing signals from the floor plate andnotochord and dorsalizing signals from the surface ec-toderm and the dorsal neural tube. This antagonisticrelationship results in a variety of transcription fac-tors, including Pax-3, Pax-6, and Pax-7, displaying re-gionally restricted expression patterns in the dorsoven-tral axis of the neural tube. Compartmentalizationwithin the rostrocaudal axis depends, at least in part,on secreted signals from the anterior visceral endoderm(Tam and Steiner, 1999), which stimulate the forma-tion of rostral structures, and on signals from the prim-itive streak and caudal paraxial mesoderm, whichstimulate the formation of caudal structures (Muhr etal., 1999). Rostrocaudal patterning is readily discern-ible through the rostrocaudally restricted expressionpatterns of Hox genes. The result of these two axes ofcompartmentalization is a grid-like pattern, in whichthe coordinates of a particular progenitor cell withinthe grid plays a prominent role in the determination ofits eventual mature neural identity (Jessell, 2001). Forexample, the dorsomedial roof of the third ventricle(DMRTV) of the postgastrulation vertebrate embryoincludes the primordia for an almost contiguous rostralto caudal string of five organs, the subfornical organ(SFO), the organum vasculosum of the lamina termi-nalis (OVLT), the diencephalic choroid plexus (DCP),the pineal gland (PG), and the subcommissural organ(SCO). These organs represent significant portals be-tween blood and neural tissue (Hofer, 1959; Johnsonand Gross, 1993), or between cerebrospinal fluid (CSF)

Grant sponsor: NICHD; Grant number: HD36871; Grant sponsor:Whitehall Foundation; Grant number: 1999-08-21.

*Correspondence to: Andrew D. Bergemann, The Lillian and HenryM. Stratton-Hans Popper, Department of Pathology, Mount SinaiSchool of Medicine, New York, NY 10029.E-mail: andrew.bergemann@mssm.edu

Received 1 October 2001; Accepted 11 February 2002DOI 10.1002/dvdy.10090Published online 3 April 2002 in Wiley InterScience (www.

interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 224:116–123 (2002)

© 2002 WILEY-LISS, INC.

and neural tissue (Schreiber et al., 1990). As a result,these organs have all been categorized as circumven-tricular organs (CVOs) (Weindl, 1973; Tsuneki, 1986),although inclusion of the DCP is controversial (John-son and Gross, 1993; Ganong, 2000). The CVOs are ofconsiderable medical importance, due to their endo-crine roles (including their control of salt and waterbalance in the body) and their roles in producing theCSF. As a result, the morphology and adult physiologyof these organs has been extensively studied. However,the number of marker genes of the primordia of thesestructures to date remains small (Duan et al., 1991;Higuchi et al., 1995; Louvi and Wassef, 2000). There-fore, we have developed a screen for genes preferen-tially expressed in the avian DMRTV compared with

the rest of the forebrain. This screen led us to identifyseveral novel interesting genes, the first of which,DNTNP (dorsal neural tube nuclear protein), encodes anuclear protein widely expressed throughout the dorsalneural tube. As we will demonstrate, DNTNP, is amember of a novel family of proteins, all of whichcontain elements resembling nuclear localization sig-nals (NLS). We will also demonstrate that the potentialNLS of DNTNP is a functional NLS, capable of target-ing green fluorescent protein (GFP) to the nucleus.

Although our screen has not produced genes specificto the DMRTV, it has identified candidate genes thatmay play important roles in its development, possiblyby contributing to the molecular definition of dorsalneural tube identity.

Fig. 1. Chicken dorsal neural tube nuclear protein (cDNTNP) expres-sion in the stage 18 chick embryo. A: Schematic representation of astage 18 chick embryo head. Purple hatching indicates dorsomedial roofof the third ventricle (DMRTV) tissue. B: Whole-mount in situ analysis ofcDNTNP expression in a stage 12 chick embryo, showing expressionthroughout the dorsal neural tube, the heart, and the somites. Bracketmarked with an S indicates the somites. Bracket marked with a P indi-cates the presomitic mesoderm, which does not express cDNTNP. C:Whole-mount in situ analysis of cDNTNP expression in a stage 18 chickembryo, showing very strong expression in dorsal neural tube, tectum,

and the cerebellar primordium, with weaker expression in the DMRTV,the telencephala, the heart, the branchial arches, the somites and thelimb buds. D: Cross-section through a stage 18 embryo, after whole-mount in situ analysis, showing strong expression in the tectum and thedorsal neural tube. E: Oblique sagittal section through a stage 18 em-bryo, after whole-mount in situ analysis, showing the high level of ex-pression in the dorsal forebrain relative to the ventral forebrain. T, tectum;DD, dorsal diencephalon; arrow, dorsal neural tube; arrowheads mark therostral and caudal boundaries of the DMRTV.

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Figure 2. (Continued.).

118 JUN ET AL.

RESULTS AND DISCUSSIONIsolation and Expression of cDNTNP

Recognizable differentiation of CVOs from surround-ing neural tissue begins around stage 18 in the chickenembryo. It is at this stage that the pineal primordiumbecomes identifiable as an evagination of the dorsaldiencephalic neuroepithelium. The DMRTV spans thedorsomedial neural tube from the caudal telencephalon(including the SFO and the OVLT), through the dien-cephalon (including the DCP and the PG), and into thepretectum of the midbrain (including the SCO) (Fig. 1A).To isolate sequences preferentially expressed in theDMRTV in the stage 17–25 chicken embryo, suppressivesubtractive polymerase chain reaction (PCR) was used toisolate sequences more highly expressed in the DMRTVrelative to the ventral diencephalon and the telencephaliccortices. The cDNA fragments generated by this ap-proach were subsequently screened for their expression

pattern in the stage 18 chicken embryo by using whole-mount RNA in situ analysis. Of the 70 clones screened forexpression, 5 showed expression patterns of interest inthe DMRTV. Of these five clones, one denoted 1-122 (datanot shown) was shown by sequencing to be a fragment ofthe previously reported Frizzled-10 gene, a knownmarker of dorsal avian neural tube (Kawakami et al.,2000). A second clone, initially denoted 1-73 displayed theinteresting expression pattern depicted in Figure 1C–E.1-73 is expressed throughout the dorsal central nervoussystem (CNS), both within the brain and the spinal neu-ral tube. Strongest expression is observed within the tec-tum and the cerebellar primordium. Within the region ofthe pretectum (the posterior border of the DMRTV), andalso the tectum, the expression of 1-73 extended morelaterally than in other regions of the CNS. Outside theneural tube, expression is also observed in the somites,the heart, the branchial arches, and the limb buds.

Fig. 2. Comparison of chicken dorsal neural tube nuclear protein(cDNTNP) and related sequences. A: An alignment of the open readingframes in cDNTNP, mDNTNP, KIAA0140, and c5ORF6, created by usingthe PILEUP program and displayed by using the PRETTYBOX programin the University of Wisconsin Genetics Computer Group package. Blackboxes indicates residues identical to the consensus for the family,whereas grey boxes indicate conservative changes. Homology regions I

and II (HR I and II) are indicated by a hatched arrow under the se-quences, the nuclear localization signal (NLS) is boxed, and the acidicdomain (AD) is marked by a dotted line. B: Phylogenetic tree of the familyconstructed by using the PHYLIP suite of programs (Felsenstein, 1989):the Protdist program was to create a distance matrix, which was thenanalyzed by using Kitsch to generate the tree. Phylodendron was used togenerate the graphical output.

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An essentially similar pattern can be observed forthe expression of 1-73 at an earlier point in develop-ment, stage 12 (Fig. 1B). At this stage, expression canalready be observed in the dorsal neural tube, as wellas the somites and the heart. Expression is notablyabsent from the presomitic mesoderm but turns onrapidly after segmentation.

Although the expression of this gene is clearly notspecific to the DMRTV, and in fact, is highest in thetectum and the cerebellar primordium, its expressionpattern indicates that it may play roles in the spec-ification of the dorsal neural tube, which is a likelyimportant step in the development of the DMRTV.

Rapid amplification of cDNA ends (RACE) experi-ments allowed us to isolate its full-length cDNAsequence with an open reading frame of 1206 bp(Genbank accession no. AF396666). As we will dem-onstrate that this protein localizes to the nucleus, wehave named this protein as cDNTNP (chicken-dorsalneural tube nuclear protein). The remaining threeclones identified by the screen are still being charac-terized and will be reported elsewhere. Like Friz-zled-10 and cDNTNP, these three clones also displaypatterns consistent with dorsal neural tube specifi-cation, rather than being restricted to the DMRTV(data not shown).

cDNTNP Belongs to a Family ofUncharacterized Proteins

The open reading frame in cDNTNP potentiallyencodes a protein of 418 amino acids in length. Itpossesses a putative NLS between residues 285 and293 (Fig. 2A). Other noteworthy features include asmall hydrophobic amino terminus (residues 1through 6) and a highly acidic carboxy-terminal do-main from residue 323 to the carboxy-terminus.Comparison of the full-length sequence of cDNTNPto the databases by using the NCBI BLAST pro-grams (Altschul et al., 1990, 1997) yielded two hu-man sequences (Fig. 2A), c5ORF6 (NP057689) andKIAA0140. c5ORF6 is a gene discovered at 5q31, whichencodes a putative nuclear protein expressed in hema-topoietic cells and tissues (Lai et al., 2000). C5ORF6was also isolated by random sequencing of cDNAs fromretinoblastoma tissue. KIAA0140 is a putative nuclearprotein (based on the presence of basic regions resem-bling NLS) derived by random sequencing of cDNAsfrom the KG-1 myeloblast cell line (Nagase et al.,1995). Amongst the related sequences in the DBESTdatabase, there are also sequences representing amouse gene (AK010537 from the RIKEN mouse ES celllibrary and AK017321 from the RIKEN mouse neonatehead library [Shibata et al., 2000]) showing a particu-larly high level of similarity to cDNTNP. We used thesemouse sequences to design primers and, thereby, re-verse transcriptase-PCR (RT-PCR) amplify the genefrom mouse cerebrocortical cDNA. The sequence of themouse gene (AF396665) encodes a protein shown inFigure 2A. At the amino acid level, the mouse sequence

shares 59% identity, KIAA0140 shares 33% identity,and c5ORF6 shares 26% identity to cDNTNP. Thisfinding indicates the mouse sequence may be an ortho-logue of cDNTNP, and for the rest of the study, it willbe referred to as mDNTNP. However, we recognizethat a more extensive analysis of the family will benecessary before this relationship can be confirmed.The closeness of the mouse and chicken sequences,however, does preclude either of the human sequencesfrom being orthologues of cDNTNP or mDNTNP (seeFig. 2B).

The family ranges from 392 amino acid residues to422 amino acid residues in length. Given the alignmentin Figure 2A, identical amino acid residues occupy 65positions in all four sequences. The positions at whichidentity is completely conserved are highly clustered attwo locations: 21 of the 65 occur between residues 112and 157 (homology region I, HR I), whereas another 28occur between residues 254 and 309 (HR II) (number-ing according to cDNTNP). The putative NLS are lo-cated within HR II. Although all four members displaya small hydrophobic amino terminus, only cDNTNPand mDNTNP display a strongly acidic carboxy-termi-nal domain.

cDNTNP Has a Functional NLS

To determine whether the putative NLS of DNTNPis indeed functional, we fused cDNTNP to green fluo-rescent protein (GFP-cDNTNP). Expression of the fu-sion protein in COS-7 cells resulted in its nuclear lo-calization (Fig. 3A). Similarly, a fusion of a 40 aminoacid fragment containing the NLS (residues 267-307)to GFP (GFP-cNLS) resulted in nuclear localization(Fig. 3C), establishing the putative NLS as a functionalnuclear localization signal. Although the nuclear dis-tribution of GFP-cNLS was uniform, that of GFP-cDNTNP frequently showed a restricted subnucleardistribution, reminiscent of nuclear bodies. Fusion ofGFP to an amino-terminal fragment (residues 1-307) ora carboxy-terminal fragment (residues 267-418), bothof which retain the putative NLS, also resulted in nu-clear localization (data not shown). As expected, GFPalone did not show nuclear localization (Fig. 3E).

EXPERIMENTAL PROCEDURESScreen for Genes Preferentially Expressed inthe DMRTV

We used the suppressive subtractive PCR approach(Diatchenko et al., 1996) to identify genes preferen-tially expressed in the DMRTV. This approach allowsthe identification of genes preferentially expressed inone particular tissue (referred to as the tracer) in com-parison to a second tissue (referred to as the driver).DMRTV tissue was dissected from 200 chicken em-bryos ranging in development from stage 17 to stage 25(as defined by Hamburger and Hamilton, 1951) andcombined for use as the tracer tissue. From the sameembryos we also dissected out telencephalic corticaltissue and ventral diencephalic tissue, which were

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Fig. 3. Nuclear localization of chicken dorsal neural tube nuclearprotein (cDNTNP). COS-7 cells transfected with vectors encoding greenfluorescent protein (GFP) fused to cDNTNP (A,B), GFP fused to a shortfragment of cDNTNP, including the putative nuclear localization signal(NLS; residues 267-307) (C,D), or GFP alone (E,F). B, D, and F are thesame cells as those in A, C, and E, respectively, viewed under a filter

appropriate for visualizing DAPI staining. White arrows indicate the nucleiof the transfected cells. G: Map of the constructs used in the analysis ofthe localization. Mutants B (residues 1-307) and C (residues 267-418)localized to nucleus (data not shown), similar to mutant D (residues267-307). HR, homology region; AD, acidic domain.

combined to form the driver tissue. Total RNA wasisolated separately from the tracer and the driver tis-sue by using the single step method (Chomczynski andSacchi, 1987; Kingston et al., 1994b). Tracer and driverRNA was converted to cDNA and amplified as outlinedin the instructions for the SMART PCR amplificationkit (Clontech) (Endege et al., 1999). Amplified cDNAswere then subjected to suppressive subtractive PCR, asoutlined in the instructions for the PCR-select subtrac-tion kit (Clontech).

Product from the suppressive subtractive PCR wascloned into pSLAX13B, a derivative of the pSLAX13vector (Hughes et al., 1987) with an altered polylinkersite. Of the resulting clones, 200 were picked andchecked for insert size. Seventy clones that displayedinserts in the range of 200 to 700 bp were subjected tosequence analysis. The expression pattern of each ofthese clones in stage 17 to 19 chicken embryos wasthen determined through whole-mount RNA in situanalysis (Tribioli et al., 1997). Digoxigenin-UTP–labelled RNA probes were generated through tran-scription of individual clones. As the library was notoriented, probes were generated for each clone by usingboth T3 and T7 RNA polymerases, and tested sepa-rately. Each treatment generated probe correspondingto the full length of the insert.

In situ analysis of cDNTNP was performed by usingprobe generated from the original 1-73 clone. 1-73 DNAwas linearized by HindIII and then used as templatefor RNA synthesis by T3 RNA polymerase. The result-ing antisense probe spanned the region from nucleotideresidue 150 to residue 438 of the sequence submitted tothe databases, which encodes residues 51 to 146 of theamino acid sequence displayed in Figure 2A. Identicalresults were obtained by using an antisense RNA tosequences in the 3� noncoding region of cDNTNP. Thesense control RNA (generated by using XbaI-linearized1-73 DNA as a substrate for T7 RNA polymerase) gaveno signal.

RACE and RT-PCR Experiments

Total RNA from chick fetal brain (embryonic day 14)and mouse cerebrocortex was extracted with TRIZOLreagent (Life Technologies, Gaithersburg, MD). RACEand RT-PCR experiments were performed according tostandard procedures. For cDNTNP, primer O06 (5�-CCACCGCTGTTGTATCGTCTCTTTG-3�) and O07 (5�-AAACCTCAACGAAAACTCGGGTCAG-3�) were usedfor 5ı́ and 3ı́ RACE, respectively. The full-lengthcDNTNP was RT-PCR amplified from total RNA byusing primer O31 (5�-GCCTCCAACACTTCTGTGTG-3�) and O32 (5�-ATGCATGGCAAAGGTAATCC-3�).The mouse transcript was amplified by using primerO28 (5�-AGAGCCCGGCCATGGTCAC-3�) and O29 (5�-TCAGCCAAAGTCAGTTGTTC-3�). All amplified prod-ucts were cloned into pBSK� vector and sequencinganalyzed.

Cellular Localization

The full-length cDNTNP cDNA was cloned as a C-terminal fusion to GFP by means of BamHI cloning inpEGFP-C1 (Clontech). Cells were transfected with 0.5�g of each plasmid construct per 3.5-cm plate, by usingthe calcium phosphate method (Graham and van derEb, 1973; Kingston et al., 1994a), and then plated oncoverslips. Forty hours after transfection, the cellswere rinsed with phosphate buffered saline and thenfixed with 4% paraformaldehyde for 5 min. Coverslipswere then mounted with VectaShield mounting me-dium containing DAPI (Vector Laboratories, Burlin-game, CA). Cell were analyzed and photographed byusing a ZEISS Axioplan microscope.

ACKNOWLEDGMENTS

We thank Edward Johnson, Jonathan Licht, FrancescaCole, Tom Lufkin, Philip Mulieri, and David Wolfe forhelpful discussions. We also thank Lucy Skrabanek, ofthe Institute of Computational Biomedicine at theMount Sinai School of Medicine, for her help with thephylogenetic analysis. This work was funded by grantsto A.D.B. from the Whitehall Foundation and the Na-tional Institute for Child Health and Development.

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