Identification, Chromosomal Assignment, and Expression Analysis of the Human Homeodomain-Containing Gene Orthopedia (OTP)

Genomics 60, 96 –104 (1999) Article ID geno.1999.5882, available online at http://www.idealibrary.com on

Identification, Chromosomal Assignment, and Expression Analysis of the Human Homeodomain-Containing Gene Orthopedia (OTP) Xu Lin,* ,1 Matthew W. State,* ,† ,1 Flora M. Vaccarino,* ,‡ John Greally,† Melanie Hass,† and James F. Leckman* ,§ ,¶,2 *Child Study Center, †Department of Genetics, ‡Department of Neurobiology, §Department of Pediatrics, and ¶Children’s Clinical Research Center, Yale University School of Medicine, New Haven, Connecticut 06520-7900 Received January 6, 1999; accepted May 21, 1999

nome in the mid-1980s (McGinnis et al., 1984a,b; Scott and Weiner, 1984), genes possessing the HD were found to be transcriptional regulators centrally involved in the development of body axes, the determination of cell fates, and the establishment of the overall patterning of the organism (Biggin and McGinnis, 1997; Lawrence and Struhl, 1996). HD genes have since been implicated in the formation of the vertebrate central nervous system (CNS) (Lumsden and Gulisano, 1997; Lumsden and Krumlauf, 1996; Rubenstein and Puelles, 1994). As in invertebrates, some HD genes identified in mammals appear to have a role in determining cell position along an axis, while others affect different characteristics of neurons, including cell proliferation, differentiation, migration, and axon path-finding (Ding et al., 1997; Salser and Kenyon, 1992; Studer et al., 1996). In an effort to identify HD genes that may be involved in the development of the human brain, we have cloned the human homologue of the murine gene Orthopedia (Otp), whose expression is confined exclusively to the CNS (Simeone et al., 1994; Genbank Accession No. Y10413). In the mouse, Otp is found in neuronal cell groups within the developing forebrain, hindbrain, and spinal cord. Specifically, Otp expression has been identified in several hypothalamic nuclei, the medial amygdala, and bed nucleus of the stria terminalus (Simeone et al., 1994). These areas are of particular interest as recent knock-out experiments have demonstrated that genes expressed in these brain regions mediate a range of maternal behaviors in rodents (Lefebvre et al., 1998; Li et al., 1999). The identification, mapping, and characterization of expression of the human HD gene will set the stage for further investigation of Otp’s possible role in the development of complex social behaviors in humans as well as in neuropsychiatric disorders such as autism (Courchesne, 1997; Insel et al., 1999).

Homeodomain (HD) genes are helix-turn-helix transcription factors that play key roles in the specification of cell fates. In the central nervous system (CNS), HD genes not only position cells along an axis, but also specify cell migration patterns and may influence axonal connectivity. In an effort to identify novel HD genes involved in the development of the human CNS, we have cloned, characterized, and mapped the human homologue of the murine HD gene Orthopedia (Otp), whose product is found in multiple cell groups within the mouse hypothalamus, amygdala, and brain stem. Human cDNA and genomic libraries were screened with probes derived from mouse Otp sequences to find the human homologue, OTP. The deduced amino acid sequence of the open reading frame of the human cDNA is 99% homologous to mouse Otp and demonstrates a high degree of conservation when compared to sea urchin and Drosophila. OTP was mapped to human chromosome 5q13.3 using radiation hybrid panel mapping and fluorescence in situ hybridization. Flanking markers were identified from YAC clones containing OTP. A single putative OTP gene product was found in 17-week human fetal brain tissue by Western blot analysis using a novel polyclonal antibody raised against a conserved 13-amino-acid sequence at the C-terminus of the OTP protein. Expression in the developing human hypothalamus was confirmed by immunohistochemistry. © 1999 Academic Press

INTRODUCTION

The homeodomain (HD) is a 180-nucleotide DNA sequence that encodes a helix-turn-helix DNA binding domain. First identified in the Drosophila ge1

The first two authors contributed equally to this report. To whom correspondence should be addressed at I-269 SHM, Child Study Center, Yale University School of Medicine, 230 South Frontage Road, P.O. Box 207900, New Haven, CT 06520-7900. Telephone: (203) 785-2511. Fax: (203) 785-7611. E-mail: james. [email protected]. 2

0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. (A) A schematic of OTP cDNA is shown. The gray box represents the coding region, the black box identifies the homeodomain, the cross-hatched region signifies the 69-bp region of homology immediately downstream of the HD region, and the horizontally striped box shows the conserved C-terminal motif. Comparison of the homeodomain region in human, Drosophila, sea urchin, and mouse homologues is shown in the box below the diagram. In the box above the diagram, the 39 domains of human and mouse OTP are compared with several paired-type homeodomain genes: Drosophila aristaless (Schneitz et al., 1993; GenBank Accession No. L08401), mouse Arx (Miura et al., 1997; GenBank Accession No. AB006103), and mouse Rax (Furukawa et al., 1997; GenBank Accession No. U73177). (B) The nucleotide sequence

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MATERIALS AND METHODS Identification of OTP cDNA and genomic DNA clones. We designed oligonucleotide primers based on the reported mouse Otp sequence (Simeone et al., 1994; Genbank Accession No. 1772994) as well as human cDNA sequence derived in our first library screenings. Primers 1.3F/1.3R included nucleotide positions 418 – 437 and the complement of positions 610 –590 (nucleotide numbers are referenced to the human cDNA sequence presented in Fig. 1B) and spanned a 189-bp region including the entire HD. Primers 5.3 F/1.4R included nucleotides 298 to 315 and the complement of 552 to 535 and spanned a 254-bp region 59 to and including the HD. Primers 3.6F/3.6R included nucleotide positions 858 to 875 and the complement of 1543 to 1523, spanning a 685-bp region 39 to the human HD. A fourth forward primer, 5.4F, was designed from human genomic DNA sequence and was used to anneal to basepairs 69 to 85. Each of the oligonucleotide primers was synthesized on an Applied Biosystems DNA synthesizer (Model 381A). Following an institutionally approved protocol, brain tissue was collected from three fetuses that were at between 10.5 and 11 weeks of gestation (David P. Bick, M.D., IVF Institute). The developmental age of these fetuses was confirmed by morphologic staging. The tissues were frozen immediately on dry ice and stored at 270°C until use. A small portion of each was examined microscopically to confirm its origin. The techniques used to prepare the total and poly(A) 1 RNA and subclone this cDNA library into a Charon BS(2) vector have been reported elsewhere (Swaroop and Xu, 1993; Swaroop and Weissman, 1988). Double-stranded DNA was prepared from the amplified phage library for use in PCR. Template DNA (100 –500 ng) from the fetal brain library was diluted into a reaction volume of 100 ml of 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.01% gelatin, a 200 mM concentration of each dNTP, and 50 pmol of primers. The buffer containing the template DNA was denatured by boiling for 10 min, after which time, 2.5 units of AmpliTaq polymerase (Cetus) was added. Mineral oil was added to cover the reaction mixture. Thirty cycles of PCR were performed with denaturation at 95°C for 1 min followed by annealing at 55°C for 1 min and extension at 70°C for 30 s using a Perkin–Elmer Cetus DNA Thermal Cycler (Murtha et al., 1991). PCR products for all primer sets were isolated by electrophoresis in 2% agarose gels. PCR products were ligated into a TA PCR vector (Invitrogen Inc.). Mini-DNA preparations from individual bacterial colonies containing recombinant plasmids were performed using standard techniques (Sambrook et al., 1989) and characterized by double-strand dideoxy sequencing using T7 polymerase and reagents from Pharmacia and United States Biochemical Co. The human fetal brain library was initially screened with a 400-bp [a- 32 P]dCTP-labeled fragment consisting of the 39 end of the mouse Otp gene, which was provided to us by Simeone and colleagues. Subsequently, the library was screened with the probes derived from the PCR primer sets described above. DNA fragments used as probes were randomly labeled with [a- 32 P]dCTP (Boehringer Mannheim Random Labeling Kit). Initial washes were carried out at low stringency (23 SSC, 42°C for 30 min). Positive clones were rescreened and washed under highstringency conditions (0.13 SSC, 68°C for 30 min). Tertiary screens were washed at high stringency (0.13 SSC, 68°C for 30 min). A human placenta genomic library (Stratagene) was also screened with the 400-bp probe provided by Simeone and colleagues. This first library screening was carried out under lowstringency conditions. Subsequent library screenings were carried

out with the full-length human OTP cDNA derived from our experiments (1 3 10 6 cpm/ml of 32 P in hybridization buffer at 42°C for 24 h). Filters were washed at 42°C for 15 min (23 SSC, 0.1% SDS) in an initial screening and then at 68°C for 30 min (0.13 SSC, 0.1% SDS) in a secondary screening. Genomic arrangement was assessed by partial restriction enzyme digestion, pulsed-field gel electrophoresis (on 1% agarose gel, at 101 mA, 6.0 V/cm for 18 h), and Southern blot. The nucleotide sequences of OTP genomic clones were determined on both strands of subfragments of DNA. Sequencing was carried out by the Keck DNA sequencing facility at Yale University. Chromosomal localization by somatic cell hybrid panel. Finemapping of OTP was undertaken using the Stanford G3 radiation hybrid panel (Research Genetics). A human-specific PCR product of 109 bp was amplified from the second intron of the OTP gene in 25-ml reactions containing 25 ng of DNA, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , a 250 mM concentration of each dNTP, 0.25 mM each primer, and 0.25 units AmpliTaq DNA polymerase (Perkin–Elmer). Initial denaturation at 94°C for 3 min was followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 90 s, and extension at 72°C for 1 min. PCR products were analyzed by electrophoresis in 2% agarose. Primer sequences were 59-GGG TCG AAC TTT GAC ACA TGC CTG-39 and 59-CGC ACT GTG CAA ACA CAC CAA ATG-39. Fluorescence in situ hybridization. Fluorescence in situ hybridization was performed as previously described (Lichter et al., 1990) with modifications. Metaphase chromosomes were prepared from human peripheral blood lymphocytes stimulated with phytohemagglutinin and harvested 72 h later from unsynchronized cultures. A 4.0-kb NotI genomic fragment of the OTP gene sequence was labeled with biotin by nick-translation. The Na-K-Cl cotransported gene (Payne et al., 1995) was used as a control and was labeled with digoxigenin by inter-Alu PCR. Sixty nanograms of OTP DNA was coprecipitated with an equivalent amount of a Na-K-Cl cotransported gene. Hybridization was carried out for 18 h under standard conditions. Detection was carried out with rhodamine-labeled anti-digoxigenen and avidin-FITC (fluorescein isothiocyanate). The chromosomes were counterstained with DAPI (4,6-diamidino-2-phenylindoledihydrochloride, 200 ng/ml). A CCD camera (PM512, Photometrics) was used to visualize fluorescent signals. Grayscale images were obtained sequentially for fluorescein, rhodamine, and DAPI with precision filter sets (Zeiss) to minimize image shifts. The grayscale images were pseudocolored and merged. Identification of flanking markers. One microliter each of culture containing CEPH-B yeast artificial chromosome (YAC) clones 728E3, 888H11, 910H4, 763C9, 765A5, 744D10, 854F5, 745A9, 805A10, 752C3, and 957A4 was grown in 50 ml of SD medium-TRP-URA (Bio 101) at 30°C for 72 h. DNA was extracted by standard methods (Sambrook et al., 1989) and was used as template for PCRs. Primers and PCR conditions were identical to those used for RH mapping noted above. Immunoblotting. A polyclonal antiserum directed against the C-terminal sequence of the OTP predicted protein product was raised against a 13-amino-acid sequence, RKALEHTVSMSFT (Fig. 1B), which is completely conserved between human and mouse OTP. Rabbits were immunized against this peptide linked to keyhole limpet hemocyanin. Sera were screened by Western blot and immunocytochemistry, using cell lines transfected with an expression vector encoding the full-length mouse Otp and appropriate controls. Sera were affinity-purified against the OTP

of OTP cDNA is shown with the deduced amino acid sequence of the open reading frame noted in single-letter code below the associated codons. The start methionine, stop codon, and polyadenylation signal are in boldface type, the homeodomain region is underlined, and the highly conserved 69-bp region is noted in italics. The boxed area at the C-terminal domain identifies the sequence used to prepare the OTP antibody. Intron/exon splice sites are noted with triangles above the corresponding nucleotide position.

CHARACTERIZATION OF Orthopedia (OTP) GENE C-terminal peptide coupled to cyanogen bromide-activated Sepharose 4b according to standard protocols (Harlow and Lane, 1988). Immunohistochemistry. Sprague–Dawley rats were purchase from a commercial breeder and were given intracardiac perfusion with 4% paraformaldehyde. The brains were removed, frozen after sucrose cryoprotection, sectioned at 20 mm on a cryostat, and stored at 220°C until use. Human fetal tissue was collected from 19-week-old fetuses (PMI, 4 – 6 h) under an institutionally approved protocol (Department of Critical Technologies, Yale University). Tissue was fixed in 4% paraformaldehyde under vacuum for 2–3 days, blocked, and frozen after sucrose cryoprotection. Tissues were sectioned at 50 mm on a microtome and stored in freezing solution (30% (w/v) sucrose, 30% (v/v) ethylene glycol, 0.001% polyvinylpyrrolidone in 0.1 M phosphate buffer pH 7.4) at 220°C until use. Immunocytochemistry was carried out essentially as described (Vaccarino et al., 1999). Briefly, sections were blocked in blocking solution (10% fetal bovine serum in PBS) for 30 min at room temperature. After sections were exposed to the OTP affinity-purified primary antiserum (1:250) overnight at 4°C in blocking solution, sections were washed three times and reacted with a biotinylated secondary antibody (1:200, Vector Laboratories) for 1 h in blocking solution. Sections were then processed using the ABC Elite kit (Vector Laboratories) according to the manufacturer’s instructions and developed with 0.025% diaminobenzine/0.003% hydrogen peroxide. After being washed with PBS, the sections were placed on gelatin-coated slides, dehydrated in an ethanol series, and mounted.

RESULTS AND DISCUSSION

cDNA and Genomic Structure of OTP Human genomic and cDNA libraries were screened with probes derived from PCR primers designed from known mouse Otp sequence and human OTP cDNA identified in our experiments. In addition, a human genomic library was screened with the full-length human OTP cDNA. Screening of the cDNA library yielded four overlapping clones. These were sequenced in both directions to yield a 2626-bp cDNA (Figs. 1A and 1B). Structural analysis of this cDNA confirmed a high degree of homology between mouse Otp and human OTP. The deduced amino acid sequences of the respective HD regions are identical (Fig. 1A). The putative products of the open reading frames differ between the two homologues at only three amino acid positions, and one of these is a conserved change. Screening of the human genomic library resulted in the identification of six overlapping clones. The longest clone contained a 16.5-kb genomic fragment. Partial restriction enzyme digestion and pulsed-field gel electrophoresis were performed to elaborate further the structure of human genomic OTP (data not shown).

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The gene was found to consist of two introns and three exons and is identical in this regard to mouse Otp. When human OTP is compared to the deduced amino acid sequences of sea urchin and Drosophila otp (Simeone et al., 1994) by BLAST search (Altschul et al., 1997), two other regions show significant homologies (Fig. 1A). The first region is immediately downstream of the HD and extends 69 bp. A second region, 36 bp in length, is found near the C-terminus of all known OTP sequences and is homologous to the motif known as the aristaless-box, which is characteristic of “paired-like” HD genes. These include aristaless (Schneitz et al., 1993), Rax (Furukawa et al., 1997), Prx-3 (van Schaick et al., 1997), Arx (Miura et al., 1997), and Cart-1 (Zhao et al., 1994). The highly conserved nature of this paired-type sequence suggests that the region may have functional significance in vivo. While this has yet to be clarified, Simeone and colleagues have demonstrated that deletion of the C-terminal region of mouse and sea urchin Otp sequences results in a threefold decrease in transactivation of a reporter construct (Simeone et al., 1994). Regional Chromosomal Mapping of OTP Chromosomal localization and identification of flanking markers were undertaken using radiation hybrid (RH) mapping, fluorescence in situ hybridization (FISH), and screening of a yeast artificial chromosome (YAC) contig. For RH mapping, PCR primers from the second intron of human OTP amplified a single 109-bp fragment using human genomic DNA as template. No PCR product was produced using identical conditions with hamster DNA as the template. Eighty-three PCRs were performed, and the amplified products were analyzed on a 4% agarose gel. Each hybrid clone was scored as amplifying the product (1), not amplifying the product (0), or equivocal (R). Results of the panel were 000 000 000 000 000 010 000 000 001 100 101 000 R00 010 000 001 011 000 000 000 000 000 000 000 000 000 000 00. These results were analyzed by two-point maximum likelihood analysis at rhserver@shgc. stanford.edu. A Lod score of 7.13 was found for the marker SHGC-7566 on chromosome 5. Data provided by the Stanford Human Genome Center located OTP between markers D5S1977 and D5S620 in the chromosomal region 5q13.3. A partial map of the human chromosome 5 showing the location of OTP is illustrated in Fig. 2B. The map

FIG. 2. Mapping human OTP. (A) Human OTP is identified (white arrows) on the proximal long arm of chromosome 5 by FISH. The idiogram next to the photograph shows the approximate location of the fluorescent signal at 5q13. A rhodamine-labeled control probe that hybridized to 5q23.3 is not shown in this photograph. (B) Relevant markers from the human chromosome region 5q13.3 are shown as hatch marks on the dark line at the top of the diagram (distances are not to scale). The lines beneath represent YACs. Boxes identify markers that are “double-linked” according to the Whitehead Institute database. Dark lines and boxes signify those YACs that were positive for OTP sequence by PCR. Dashed lines and open boxes signify YACs that did not amplify OTP sequence. YAC identifiers are listed in the right-hand column. All data regarding the location of markers on YACs are taken from the Whitehead Institute for Biomedical Research/MIT Center for Genome Research (http://www.genome.wi.mit.edu/).

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FIG. 4. OTP immunoreactivity in the rat hypothalamus. Frozen coronal sections from the hypothalamic region of rats at postnatal day 19 were reacted with the OTP affinity-purified antiserum. PVN, paraventricular nucleus, SON, supraoptic nucleus of the hypothalamus. Scale bar, 100 mm. FIG. 5. OTP immunoreactivity in the human hypothalamus. Frozen coronal sections from the hypothalamic region of a human fetus at 19 weeks of gestation were reacted with the OTP affinity-purified antiserum. Dorsal is on top, and medial is on the left. Arrows indicate positive nuclei. (a) and (b) supraoptic nucleus; (c) paraventricular region. Scale bar, 400 mm in (a) and 100 mm in (b) and (c).

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FIG. 3. Expression of the OTP gene product in human embryonic brain tissue. Extracts of whole brain (lanes 1, 3) and brainstem (lanes 2, 4) from human embryos at 17 weeks of gestation were fractionated by SDS–PAGE (50 mg per lane) and transferred to a PVDF membrane (Millipore). The blot was probed with the OTP affinity-purified antiserum (1:250 dilution) and developed using chemoluminescence for detection. In lanes 1 and 2, the antiserum was used without preabsorption, and in lanes 3 and 4, the antiserum was preincubated for 1 h in a 10-fold excess of the OTP C-terminal peptide.

was constructed based on information obtained from the Whitehead Institute. The primers used for RH mapping were used to screen the identified YACs from the CEPH B library (Fig. 2B). Initially six YAC clones spanning the interval between D5S1977 and D5S620 were evaluated. Only YAC 910H4 was positive for OTP sequence by PCR. Subsequently, six additional YACs were evaluated. Clones 805A10, 957A4, 745A9, and 752C3 were found to contain OTP sequence. All of the positively identified YAC clones also contained the marker D5S1962, which is therefore likely to be closest to the human OTP locus. Markers IB3702 and WI-6272 flank the OTP locus. Four of five YAC clones found to contain OTP sequence also contained the marker IB3702. YACs 888H11 and 854F5, which contain the marker WI-6272, did not show OTP sequence (Fig. 2B). FISH analysis confirmed the chromosomal localization (Fig. 2A). Twenty-four metaphase spreads were visualized. In each case, FITC signals were seen on chromosome band 5q13. The chromosomal assignment was confirmed by cohybridization with the KEF gene that has previously been mapped to 5q23.3 (data not shown). These mapping results are consistent with the mapping of murine Otp to a syntenic region of mouse chromosome 13 (Wang and Lufkin, 1997). Expression of OTP To detect the expression of the OTP gene product in human tissue, we generated a polyclonal antiserum

directed against the C-terminal sequence of the OTP predicted protein product. The OTP antibodies labeled a band of approximately 50 kDa in Western blots of rat and human embryonic brain tissue (Fig. 3). This band was not observed upon preabsorption of the OTP antibodies with the OTP C-terminal peptide used to immunize the rabbits (Fig. 3). Immunocytochemistry using these OTP antibodies yielded staining patterns in mouse and rat diencephalon and telencephalon corresponding to the expression of Otp mRNA in these regions (data not shown). The predicted size of mouse and human OTP is 36 kDa, whereas we detected a species of approximately 50 kDa by Western blot both in human fetal brain and in rodent brain tissue. A single band of apparent molecular mass of 48 –50 kDa was also detected in cell lines transfected with mouse Otp expression vector (data not shown). These data and the preabsorption experiment suggest that the 50-kDa band is, in fact, OTP. The difference between our observations and the expected size of the protein may be the result of anomalous migration, which is sometimes observed with homeodomain proteins on SDS gels (Lin et al., 1996; Lin et al., unpublished data). Immunocytochemistry using these OTP antibodies yielded staining patterns in mouse and rat diencephalon and telencephalon corresponding to the expression of Otp mRNA in these regions (Fig. 4). Specifically, intense staining was observed in the paraventricular and supraoptic regions of the hypothalamus and the medial amygdala. Immunostaining of fetal human tissue at 19 weeks of gestation, revealed a highly restricted pattern of expression within the forebrain. OTP-like immunoreactivity was present within the hypothalamus, particularly in the supraoptic region, consistent with the localization of OTP in rodent tissue (Figs. 4 and 5). No staining was observed in the cerebral cortex, subcortical white matter, and other developing fiber tracts. OTP staining was prominent in cell nuclei, although occasionally faint cytoplasmic staining was also observed. The nuclear morphology of the great majority of OTP-immunopositive cells was elongated, consistent with that of migratory cells. Many of these migratory-like cells were located in a region dorsolateral to the supraoptic nucleus, between the hypothalamus and the prospective amygdaloid region (Fig. 5). Cells within the developing supraoptic nucleus had large oval nuclei, faint staining in the cytoplasm, and no staining in their processes. Staining was also visualized within the paraventricular region, consistent with the localization of the paraventricular nucleus. In summary, the human OTP gene product is highly conserved with respect to both sequence and regional location within the forebrain. As in the mouse, human OTP expression is restricted to a group of interconnected cells in the hypothalamus, amygdala, and related limbic regions. These cell groups define a distinct circuit in the brain that may be involved in the control of emotional and neuroendocrine functions in humans.

CHARACTERIZATION OF Orthopedia (OTP) GENE

Our hypothesis that human OTP would be expressed in human brain and, specifically, in the human hypothalamus has been confirmed by our data. The identification of the human homologue of this CNS-specific HD gene now sets the stage for further studies of OTP expression in humans and its involvement in both normal and pathological brain development. ACKNOWLEDGMENTS This work was supported in part by the Korzac Foundation and a grant from the National Institute of Mental Health (MH49351). The authors thank M. Ding for her help in the characterization of the OTP antiserum and A. Simeone for providing the murine Otp probe as well as the Otp expression vectors. We extend our appreciation to D. Ward, P. Burbach, M. Biggin, E. Burnett, M. Murtha, and P. J. Lombroso for their comments during the preparation of the manuscript. We also thank Drs. A Swaroop, D. P. Bick, R. Robins, K. Kagan-Hallet, and J. McGill for their roles in collecting and preparing the fetal brain library used in this study. C. Howe assisted in the collection and preparation of the human fetal tissue used in the expression study.

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