Identification of Genes Downstream of Pax6 in the Mouse Lens Using cDNA Microarrays
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m110531200
ISSN1083-351X
AutoresBharesh K. Chauhan, Nathan Reed, Weijia Zhang, Melinda K. Duncan, Manfred W. Kilimann, Aleš Cvekl,
Tópico(s)Photochromic and Fluorescence Chemistry
ResumoPax6 is a transcription factor that regulates the development of the visual, olfactory, and central nervous systems, pituitary, and pancreas. Pax6 is required for induction, growth, and maintenance of the lens; however, few direct Pax6 target genes are known. This study was designed to identify batteries of differentially expressed genes in three related systems: 8-week old Pax6 heterozygous lenses, 8-week old Pax6 heterozygous eyes, and transgenic lenses overexpressing PAX6(5a), using high throughput cDNA microarrays containing about 9700 genes. Initially, we obtained almost 400 differentially expressed genes in lenses from mice heterozygous for a Pax6 deletion, suggesting that Pax6 haploinsufficiency causes global changes in the lens transcriptome. Comparisons between the three sets of analyses revealed that paralemmin, molybdopterin synthase sulfurylase, Tel6 oncogene (ETV6), a cleavage-specific factor (Cpsf1) and tangerin A were abnormally expressed in all three experimental models. Semiquantitative reverse transcription (RT)-PCR analysis confirmed that all five of these genes were differentially expressed in Pax-6 heterozygous and Pax6(5a) transgenic lenses. Western blotting and immunohistochemistry demonstrated that paralemmin is found at high levels in the adult lens and confirmed its down-regulation in the Pax6(5a)-transgenic lenses. Collectively, our data provide insights into the genetic programs regulated by Pax6 in the lens. Pax6 is a transcription factor that regulates the development of the visual, olfactory, and central nervous systems, pituitary, and pancreas. Pax6 is required for induction, growth, and maintenance of the lens; however, few direct Pax6 target genes are known. This study was designed to identify batteries of differentially expressed genes in three related systems: 8-week old Pax6 heterozygous lenses, 8-week old Pax6 heterozygous eyes, and transgenic lenses overexpressing PAX6(5a), using high throughput cDNA microarrays containing about 9700 genes. Initially, we obtained almost 400 differentially expressed genes in lenses from mice heterozygous for a Pax6 deletion, suggesting that Pax6 haploinsufficiency causes global changes in the lens transcriptome. Comparisons between the three sets of analyses revealed that paralemmin, molybdopterin synthase sulfurylase, Tel6 oncogene (ETV6), a cleavage-specific factor (Cpsf1) and tangerin A were abnormally expressed in all three experimental models. Semiquantitative reverse transcription (RT)-PCR analysis confirmed that all five of these genes were differentially expressed in Pax-6 heterozygous and Pax6(5a) transgenic lenses. Western blotting and immunohistochemistry demonstrated that paralemmin is found at high levels in the adult lens and confirmed its down-regulation in the Pax6(5a)-transgenic lenses. Collectively, our data provide insights into the genetic programs regulated by Pax6 in the lens. Pax6 is among the most widely studied transcription factors because of its participation in the organogenesis of the eye, brain, head, and pancreas (1.Glaser T. Walton D.S. Cai J. Epstein J.A. Jepeal L. Maas RL. Wiggs J. Molecular Genetics of Ocular Disease. John Wiley and Sons, Inc., New York1995: 55-81Google Scholar, 2.Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-532Crossref PubMed Scopus (390) Google Scholar, 3.Jean D. Ewan K. Gruss P. Mech. Dev. 1998; 76: 3-18Crossref PubMed Scopus (135) Google Scholar, 4.Mansouri A. St-Onge L. Gruss P. Trends Endocrinol. Metab. 1999; 10: 164-167Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The essential role of Pax6 in early eye induction is conserved throughout the evolution of multicellular animals with ectopic expression able to direct conversion of wing imaginal disks to eyes in Drosophila melanogaster (2.Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-532Crossref PubMed Scopus (390) Google Scholar) and head ectoderm to lenses in Xenopus laevis (5.Altmann C.R. Chow R.L. Lang R.A. Hemmati-Brivalou A. Dev. Biol. 1997; 185: 119-123Crossref PubMed Scopus (146) Google Scholar). In the vertebrate eye, Pax6 is required for lens placode formation, growth of the lens (6.Van Raamsdonk C.D. Tilghman S.M. Development. 2000; 127: 5439-5448Crossref PubMed Google Scholar), correct placement of a single retina in the eye (7.Ashery-Padan R. Marquardt T. Zhou X Gruss P. Genes Dev. 2000; 14: 2701-2711Crossref PubMed Scopus (459) Google Scholar), formation of the iris, maintenance of the corneal epithelium, and fate of retinal progenitor cells (8.Marquardt T. Ashery-Padan R. Andrejewski N. Scardigli R. Guillemot F. Gruss P. Cell. 2001; 105: 43-55Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar). The diverse functions of Pax6 appear to originate from both the complex regulatory mechanisms controlling the tissue-specific transcription and splicing of the Pax6 mRNA as well as its ability to participate in multiple molecular interactions. A prevailing form of Pax6 in mouse embryos contains two DNA-binding domains, the paired domain and homeodomain (HD), 1The abbreviations used are: HDhomeodomainRTreverse transcriptionESTexpressed sequence tagMOCS3molybdopterin synthase sulfurylase 1The abbreviations used are: HDhomeodomainRTreverse transcriptionESTexpressed sequence tagMOCS3molybdopterin synthase sulfurylase which can interact both independently and cooperatively with DNA, whereas the C terminus comprises the transcriptional activation domain (9.Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar, 10.Glaser T. Jepeal L. Edwards J.G. Young S.R. Favor J. Maas R.L. Nat. Genet. 1994; 7: 463-471Crossref PubMed Scopus (598) Google Scholar). The paired domain contains two subdomains, PAI and RED, each of them capable of binding independently to DNA (9.Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar). A splice variant, Pax6(5a), has an additional 14 amino acids inserted into the PAI subdomain. This results in its recognition of only a subset of Pax6 binding sites (11.Epstein J.A. Glaser T. Cai L. Jepeal L Maas R.L. Genes Dev. 1994; 8: 2022-2034Crossref PubMed Scopus (317) Google Scholar, 12.Kozmik Z. Czerny T. Busslinger M. EMBO J. 1997; 16: 6793-6803Crossref PubMed Scopus (129) Google Scholar, 13.Yamaguchi Y. Sawada J. Yamada M. Handa H. Azuma N. Genes Cells. 1997; 2: 255-261Crossref PubMed Scopus (40) Google Scholar, 14.Duncan M.K. Kozmik Z. Cveklova K. Piatigorsky J. Cvekl A. J. Cell Sci. 2000; 113: 3173-3185Crossref PubMed Google Scholar). Recent evidence suggests that Pax6 function is further modulated by interactions of its homeodomain with a diverse set of proteins, including the homeodomain-containing proteins Six3, Prox1, and Lhx2 (15.Mikkola I. Bruun J.-A. Holm T. Johansen T. J. Biol. Chem. 2001; 276: 4109-4118Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and the transcription factors TFIID and pRb (16.Cvekl A. Kashanchi F. Brady J.N. Piatigorsky J. Invest. Ophthalmol. Vis. Sci. 1999; 40: 1343-1350PubMed Google Scholar). Pax6 also physically interacts with c-Maf/Maf A (17.Planque N. Leconte L. Coquelle F.M. Benkhelifa S. Martin P. Felder-Schmittbuhl M.-P. Saule S. J. Biol. Chem. 2001; 276: 35751-35760Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and MitF (microphthalmia) (18.Planque N. Leconte L. Coquelle F.M. Martin P. Saule S. J. Biol. Chem. 2001; 276: 29330-29337Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), two important transcription factors controlling lens differentiation, and retinal development, respectively. homeodomain reverse transcription expressed sequence tag molybdopterin synthase sulfurylase homeodomain reverse transcription expressed sequence tag molybdopterin synthase sulfurylase While Pax6 is clearly a central player in many developmental processes, relatively few genes have been shown to be directly regulated by Pax6. In Drosophila, Pax6/ey directly regulates the transcription of rhodopsins (19.Papatsenko D. Nazina A. Desplan C. Mech. Dev. 2001; 101: 143-153Crossref PubMed Scopus (55) Google Scholar) and sine oculis (20.Niimi T. Seimiya M. Kloter U. Flister S. Gehring W.J. Development. 1999; 126: 2253-2260Crossref PubMed Google Scholar). In vertebrates, Pax6 directly affects expression of Pax2 in the developing optic cup and stalk (21.Schwarz M. Cecconi F. Bernier G. Andrejewski N. Kammandel B. Wagner M. Gruss P. Development. 2000; 127: 4325-4334Crossref PubMed Google Scholar). Genetic evidence suggests that the genes for the eye development regulators Eya1 and -2 (22.Xu P.X. Woo I. Her H. Beier D.R. Maas R.L. Development. 1997; 124: 219-231Crossref PubMed Google Scholar), Sox-2 (7.Ashery-Padan R. Marquardt T. Zhou X Gruss P. Genes Dev. 2000; 14: 2701-2711Crossref PubMed Scopus (459) Google Scholar), and c-Maf (23.Sakai M. Serria M.S. Ikeda H. Yoshida K. Imaki J. Nishi S. Nucleic Acids Res. 2001; 29: 1228-1237Crossref PubMed Scopus (65) Google Scholar) are also direct targets. In addition to these developmental regulators, Pax6 can directly regulate the insulin, glucagon, and somatostatin genes expressed in the pancreas (22.Xu P.X. Woo I. Her H. Beier D.R. Maas R.L. Development. 1997; 124: 219-231Crossref PubMed Google Scholar); L1-CAM expression in the brain (25.Meech R. Kallunki P. Edelman G.M. Jones F.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2420-2425Crossref PubMed Scopus (76) Google Scholar); keratin K12 (26.Shiraishi A. Converse R.L. Liu C.Y. Zhou F. Kao C.W.C. Kao W.W.Y. Invest. Ophthalmol. Vis. Sci. 1998; 39: 2554-2561PubMed Google Scholar) and gelatinase B (27.Sivak J.M. Mohan R. Rinehart W.B. Xu P.X. Maas R.L. Fini M.E. Dev. Biol. 2000; 222: 41-54Crossref PubMed Scopus (81) Google Scholar) expression in the cornea; and αA-, αB-, δ1-, βB1- and ζ-crystallin expression in the lens (28.Cvekl A. Piatigorsky J. Bioessays. 1996; 18: 621-630Crossref PubMed Scopus (247) Google Scholar, 29.Kondoh H. Curr. Opin. Genet. Dev. 1999; 9: 301-308Crossref PubMed Scopus (58) Google Scholar, 30.Duncan M.K. Haynes J.I. Cvekl A. Piatigorsky J. Mol. Cell. Biol. 1998; 18: 5579-5586Crossref PubMed Google Scholar). Although the mechanism of Pax6 function has not been studied in detail in many of these cases, it appears that it can function both as a transcriptional activator and repressor (30.Duncan M.K. Haynes J.I. Cvekl A. Piatigorsky J. Mol. Cell. Biol. 1998; 18: 5579-5586Crossref PubMed Google Scholar) in in vitro assays. cDNA microarray technology has been developed to decipher the complex genetic networks altered in response to environmental insults and disease (31.Lockhart D.J. Winzeler E.A. Nature. 2000; 405: 827-836Crossref PubMed Scopus (1691) Google Scholar, 32.Duggan D.J. Bittner M. Chen Y. Meltzer P. Trent J.M. Nat. Genet. 1999; 21S: 11-14Google Scholar, 33.Livesey F.J. Furukawa T. Steffen M.A. Church G.M. Cepko C.L. Curr. Biol. 2000; 10: 301-310Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Here, this technology is used to study Pax6 function by determining which genes are affected by both Pax6 haploinsufficiency in the eye (1.Glaser T. Walton D.S. Cai J. Epstein J.A. Jepeal L. Maas RL. Wiggs J. Molecular Genetics of Ocular Disease. John Wiley and Sons, Inc., New York1995: 55-81Google Scholar, 6.Van Raamsdonk C.D. Tilghman S.M. Development. 2000; 127: 5439-5448Crossref PubMed Google Scholar, 7.Ashery-Padan R. Marquardt T. Zhou X Gruss P. Genes Dev. 2000; 14: 2701-2711Crossref PubMed Scopus (459) Google Scholar, 8.Marquardt T. Ashery-Padan R. Andrejewski N. Scardigli R. Guillemot F. Gruss P. Cell. 2001; 105: 43-55Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar) and Pax6(5a) overexpression (14.Duncan M.K. Kozmik Z. Cveklova K. Piatigorsky J. Cvekl A. J. Cell Sci. 2000; 113: 3173-3185Crossref PubMed Google Scholar) in the lens. Our studies demonstrate the usefulness of microarray analysis for the analysis of gene expression in pathological conditions and give some insight into the function of Pax6 in the mature lens. NMRI mice heterozygous for a Pax6 knockout/lacZ knock-in allele were generously provided by Dr. Peter Gruss (Max-Planck-Institute of Biophysical Chemistry, Gottingen, Germany) (34.St-Onge L. Sosa-Peneda B. Chowdhury K. Gruss P. Nature. 1997; 387: 406-409Crossref PubMed Scopus (663) Google Scholar), while wild type NMRI mice were obtained from Charles River Laboratories (L'Arbresle, France). FVB/N mice overexpressing Pax6(5a) in lens fiber cells under the control of the mouse αA-crystallin promoter and wild type strain matched controls were described previously (14.Duncan M.K. Kozmik Z. Cveklova K. Piatigorsky J. Cvekl A. J. Cell Sci. 2000; 113: 3173-3185Crossref PubMed Google Scholar). Lenses and eyes from which lenses were surgically removed were isolated from 8-week-old Pax6 heterozygous and wild type mice and stored in RNAlater (Ambion, Woodlands, TX) until RNA isolations were performed using the Totally RNA kit (Ambion). The genotype of Pax6 heterozygous lenses was confirmed by assaying the expression of Pax6 and lac Z using RT-PCR using primers designed to amplify Pax6 (5′-TTT AAC CAA GGG CGG TGA GCA G-3′ and 5′-TCT CGG ATT TCC CAA GCA AAG ATG-3′) and lacZ mRNAs (5′-GTC AGG TCA TGG ATG AGC AG-3′ and 5′-CAC TAC GCG TAC TGT GAG C-3′) employing the One Step RT-PCR system (Invitrogen). The initial RT step was conducted at 50 °C for 30 min, and amplifications were conducted at the annealing temperature of 58 °C. Lenses were isolated from 3- and 8-week mice overexpressing PAX6(5a) in lens fiber cells and strain-matched controls as described (14.Duncan M.K. Kozmik Z. Cveklova K. Piatigorsky J. Cvekl A. J. Cell Sci. 2000; 113: 3173-3185Crossref PubMed Google Scholar), and RNA was immediately prepared using the SV Total RNA Isolation System (Promega, Madison, WI). cDNAs were generated using 2–5 μg of total RNA and indirectly labeled with Cy3- and Cy5-specific dendrimers, employing the 3DNA detection system from Genisphere, Inc. (Montvale, NJ) (35.Stears R.L. Getts R.C. Gullans S.R. Physiol. Genomics. 2000; 3: 93-99Crossref PubMed Scopus (189) Google Scholar) according to the manufacturer's protocol. Glass slide microarrays containing about 9700 mouse sequence verified genes were described elsewhere (36.Cheung V.G. Morley M. Aguilar F. Massimi A. Kucherlapati R. Childs G. Nat. Genet. 1999; 21S: 15-19Crossref Scopus (565) Google Scholar). The hybridizations were performed at 50 °C, with three subsequent washes of the slides performed in 2× SSC, 0.2% SDS; 2× SSC; and 0.2× SSC buffers. The chips were scanned using the GenePix 4000A scanner (Axon Instruments, Union City, CA), and primary data were analyzed using the Genepix 3.02 software. Each experiment was conducted in triplicate. Control self-hybridizations were performed using wild type RNA to determine S.D. values that were used to determine the eventual cut-off values. Primary data were flagged using four default parameters set in the Genepix 3.0 program. Intensity data for both channels were normalized by the widely used global intensity normalization method (37.Zavadil J. Bitzer M. Liang D. Yang Y.-C. Massimi A. Kneitz S. Piek E. Bottinger E.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6686-6691Crossref PubMed Scopus (445) Google Scholar). The intensity of each spot in each channel was adjusted by subtracting the local background from the observed intensity (I′ij =Iij − Bij, where I′ij, Iij, and Bij are the adjusted intensity, observed intensity, and background for the jth gene (j = 1, 2, … n) in the ith channel (i = 1, 2), respectively), and then subjected to log transformation (ln(I′ij)). The overall intensity for each channel was calculated by taking the power of the average of the log of the adjusted intensity for all genes (ICh1 =eln(I′1j)/n, and ICh2 =eln( I′2 j)/n, where n is the number of genes, and ICh1 and ICh2 are the overall intensity for channel 1 and channel 2, respectively). The intensities for both channels were therefore balanced by multiplying the adjusted intensity of each spot in channel 2 by the ratio of the overall intensity in channel 1 over that in channel 2 (r =ICh1/ICh2). Means and S.D. values were calculated for those genes with no more than one flagged data point. For normalized data tables, see the Supplemental Material. Genes were classified into 12 functional groups (38.Gerstein M. Jansen R. Curr. Opin. Struct. Biol. 2000; 10: 574-584Crossref PubMed Scopus (58) Google Scholar) using annotations from the Swissprot data base (available on the World Wide Web at ca.expasy.org/sprot). The tangerin A was identified from ESTAA217475 using the Gencarta data base (Compugen Ltd., Tel Aviv, Israel). All transcripts studied were reverse-transcribed and amplified using the One Step RT-PCR system (Invitrogen). The initial reverse transcriptase step was conducted at 50 °C for 30 min. The annealing temperatures used for individual experiments are indicated in Table I. All amplifications shown here were performed at 29 cycles. All primers used in this study (Table I) were designed to cross intron-exon boundaries and were tested in the absence of reverse transcriptase. Control reactions were performed initially to ensure linearity of amplification over concentrations of total RNA ranging from 5 to 100 ng.Table IPrimers used in this studyPrimer nameSequence (5′–3′)Product lengthAnnealing temperaturebp°CArgininosuccinate synthetaseGAAGCTTGGGGCCAAAAAGG10653ATAGCGGTCCTCGTAGAGTGUridine monophosphate kinaseTGGAAGTGCCCTTTGCTGTC14155CAGTTCAACAAAACCAGCCCAGUbiquitin-conjugating enzymeACTAGACACCCGACCCTTAC13654.9AAAGGATGAGGCTGTGGTGGCoq7/clk1GAAGAGGATTATCCAGGCCG12550.3CCTCCTCCACAAATATCACTCPax6/Pax6(a)CGGCAGAAGATCGTAGAG289/33156GATGACACACTGGGTATGParalemminCGACGAGGACATGAGGAAAC14055.3CTGAATTCTCCTTTGAGGCAGCTangerin AAGTAACCCTGGTGGACAAG29254.8GAGAAGAGGAAATGGGGGAGMOCS3CCCGGTGTATGTGATTTGC14954.6TCCCATCAATTCTGACGGCEtv6TGTGCAGCAGAGACATCTCC14054.8CCGGCAATACACTCCTTACAGCspf1TTGGACAGCCCACTACACAAGG10055CTTCCCAGAGCAACCAACAACAC Open table in a new tab Lenses were dissected from 6-week-old Pax6(5a) transgenic and wild type litter mates and homogenized in radioimmune precipitation buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.575 mm phenylmethylsulfonyl fluoride, 45 μg/ml aprotinin, 1 mm sodium orthovanadate). Supernatants were collected following two spins at 10,000 × g. Protein concentrations were immediately determined using Bio-Rad DC protein assay (Bio-Rad), and 56 μg of protein were loaded on each lane of a 10% discontinuous SDS-PAGE gel. The protein was transferred to nitrocellulose and incubated with a 1:2000 dilution of anti-paralemmin rabbit crude serum (39.Kutzleb C. Sanders G. Yamamoto R. Wang X. Lichte B. Petrash-Parwez E. Kilimann M.W. J. Cell Biol. 1998; 143: 795-813Crossref PubMed Scopus (61) Google Scholar). Bound antibodies were detected with 1:2000 horseradish peroxidase-linked anti-rabbit IgG (New England BioLabs). Blots were developed using LumiGLO (Cell Signaling Technologies, Beverly, MA) and exposed to film. Mouse eyes were enucleated and embedded in Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC), and 16-μm frozen sections were prepared. Sections were then fixed in 1:1 acetone/methanol for 10 min at −20 °C and blocked with 1% bovine serum albumin/phosphate-buffered saline for 1 h at RT. Paralemmin and preimmune paralemmin primary antibodies (39.Kutzleb C. Sanders G. Yamamoto R. Wang X. Lichte B. Petrash-Parwez E. Kilimann M.W. J. Cell Biol. 1998; 143: 795-813Crossref PubMed Scopus (61) Google Scholar) were prepared in 1% bovine serum albumin/phosphate-buffered saline at dilutions of 1:150. The bound primary antibodies were visualized following incubation with anti-rabbit IgG conjugated with Alexa Fluor 568 (1:50 dilution in 1% bovine serum albumin/phosphate-buffered saline; Molecular Probes, Inc., Eugene, OR). The cell nuclei were detected by counterstaining with SYTO-13 (1:1000 dilution; Molecular Probes). Confocal microscopy was performed on a Zeiss LSM 510 confocal microscope (Carl Zeiss, Königsallee, Göttingen, Germany) configured with an argon/krypton laser (488- and 568-nm excitation lines). A more detailed protocol may be found in Reed et al.(40.Reed N.A. Oh D.-J. Czymmek K.J. Duncan M.K. J. Immunol. Methods. 2001; 253: 243-252Crossref PubMed Scopus (51) Google Scholar). Initially, lens RNA obtained from 8-week-old NMRI mice was labeled with either Cy5- or Cy3-labeled dendrimers (35.Stears R.L. Getts R.C. Gullans S.R. Physiol. Genomics. 2000; 3: 93-99Crossref PubMed Scopus (189) Google Scholar) and self-hybridized to cDNA microarrays containing about 9700 sequence-verified genes (36.Cheung V.G. Morley M. Aguilar F. Massimi A. Kucherlapati R. Childs G. Nat. Genet. 1999; 21S: 15-19Crossref Scopus (565) Google Scholar) to determine the S.D. value of the hybridization ratios (Cy5/Cy3). Since the S.D. obtained from three independent hybridizations was 0.28–0.31, expression ratios more than 1.60 for up-regulated and less than 0.63 for down-regulated genes are statistically significant, since they represent values that differ by two S.D. values. Genes differentially expressed in Pax6 heterozygous lenses as compared with normal lenses were determined by labeling cDNAs from the two samples with Cy3- and Cy5-specific dendrimers before simultaneously probing onto the cDNA microarrays described above. A representative scatter plot from one experiment showing the distribution of hybridization signals generated using GeneSpring 3.02 software (Silicon Genetics, San Carlos, CA) is shown in Fig. 1A. To demonstrate the reproducibility of data using the 3DNA labeling technology (35.Stears R.L. Getts R.C. Gullans S.R. Physiol. Genomics. 2000; 3: 93-99Crossref PubMed Scopus (189) Google Scholar), we randomly selected six genes (GenBankTM accession numbersAA387340, AA120030, AA445775, AA238399, AA260490, and AA000249), and their ratios of expression from triplicate microarrays are given in Fig. 1B. This is the first report, to our knowledge, using the 3DNA detection system (Genisphere) and poly-l-lysine-coated slides used to print the microarrays, allowing one to work with 2–5 μg of total RNA without any mRNA amplification step (33.Livesey F.J. Furukawa T. Steffen M.A. Church G.M. Cepko C.L. Curr. Biol. 2000; 10: 301-310Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). In addition, the standard deviation of the control experiment, 0.28–0.31, was comparable with direct incorporation methods employing Cy3′ and Cy5′ modified UTP, which typically yielded values between 0.19 and 0.21. 2B. K. Chauhan and A. Cvekl, unpublished data. Normalized data tables can be obtained on the World Wide Web at www.aecom.yu.edu/thecvekllab. Some of the data were flagged due to the unacceptable signal intensity above background intensity (i.e. if the signal intensity above background intensity was less than 100, then the spot would be flagged). This resulted in the identification of more than 400 differentially expressed genes; the vast majority of them were down-regulated, consistent with Pax6 roles as an activator of transcription. From these data, three lists of genes were generated. The first list, shown in Table II, includes genes with known functions classified into 12 subcategories (38.Gerstein M. Jansen R. Curr. Opin. Struct. Biol. 2000; 10: 574-584Crossref PubMed Scopus (58) Google Scholar), flagged no more than once, and expressed in Pax6 heterozygous lenses at reduced levels up to a factor of 0.63 and up-regulated genes by a factor of at least 1.6. When a single flag was found, we included the data if mean and median values were similar. The second list, shown in Table III, includes known genes that could not be classified into one of the 11 functional categories. The third list, shown in Table IV, includes ESTs showing strong and moderate similarities with genes deposited in public data bases and contained no more than one flagged value, as described above. Two genes down-regulated in Pax6 heterozygous lenses and relevant to known lens biology are homeodomain-containing transcription factor Pitx3 and structural βA4-crystallin (41.Duncan M.K. Haynes II, J.I. Piatigorsky J. Gene (Amst.). 1995; 162: 189-196Crossref PubMed Scopus (19) Google Scholar).Table IIClassified list of known genes abnormally expressed in Pax6 heterozygous lensesGeneDescriptionGenBank™ accession no.ChangeCell growth, division, and DNA synthesis-fold Akt2Thymoma viral proto-oncogene 2W82557−2.1 Ccne2Cyclin E 2AA414293−7.7 Lig1Ligase I, DNA, ATP-dependentW66626−7.8 Ptpn16Protein-tyrosine phosphatase, nonreceptor type 16AA125367−7.3 Rab19RAB19, member rasoncogene familyAA118762−1.6 StmnStathminAA265396−1.7 Rfc1Replication factor C, 140 kDaAA011737−2.1Cell rescue, defense, and death Bcl10B-cell leukemia/lymphoma 10 (CARD-containing proapoptotic protein)W47752−2.2 Brf1Butyrate response factor 1AA060205−1.7 F9Coagulation factor IXAA209011−3.6 H2-ObHistocompatibility 2, O region β locusAA145469−2.4 Ms4a2Membrane-spanning 4 domains, subfamily A, member 2AA183371−2.0Cellular organization mACF7Mouse actin cross-linking factor, neural isoform 2W65621−1.9 Adam9A disintegrin and metalloproteinase domain 9 (meltrin γ)AA210306−3.3 Cappa1Capping protein α 1AA414612−4.8 Cdh3Cadherin 3W12889−2.0 Cryba4Crystallin, β A4W82104−1.6 DsnDestrinW17549−1.9 ImgIntegral membrane glycoproteinW83922−1.5 PalmParalemminY14771−3.6 Plxn2Plexin 2AA511430−2.5 Plxn3Plexin 3W76838−1.8 Pfn2Profilin 2AA139628−8.9 Smoc1Secreted modular calcium-binding protein 1AA000223−2.3 Spnb3β-Spectrin 3AA049581−1.9 Thbs4Thrombospondin 4AA003452−1.9 Tubb5Tubulin, β 5W16254−1.5Energy Slc25a10Mitochondrial solute carrier 25a10 (adenine nucleotide translocator)W66635−1.6Intracellular transport Nxt1-pendingNTF2-related export protein 1W13971−3.9 PMCA1Plasma membrane calcium ATPase isoform 1 (Rattus norvegicus)AA125425+1.8 Sec23aSEC23A (Saccharomyces cerevisiae)AA213082−3.1Ionic homeostasis Clcn7Chloride channel 7W08488−1.7Metabolism AsnsAsparagine synthetaseW29492−1.6 DhodDihydroorotate dehydrogenaseAA238399−2.6 DRP3Dihydropyrimidinase-related protein 3W18828−1.8 GnmtGlycine N-methyltransferaseW83078−5.0 Hmgcs23-Hydroxy-3-methylglutaryl-coenzyme A synthase 2AA030116−2.9 Hsd3b1Hydroxysteroid dehydrogenase-1, Δ〈5〉-3-βAA028760−4.5 Mocs3Molybdopterin synthase sulfurylase (Homo sapiens)W99918−4.2 Mor2Malate dehydrogenase, solubleW13686−2.2 PahxPhytanoyl-CoA α-hydroxylaseW82212−1.8 Peci-pendingPeroxisomal Δ3, Δ2-enoyl-coenzyme A isomeraseAA030780−2.2 PycsPyrroline-5-carboxylate synthetaseW41878−1.6Protein synthesis and degradation Cpsf1Cleavage and polyadenylation-specific factorW41928−2.2 Eif1aEukaryotic translation initiation factor 1AW41459−1.9 Eif2s3xEukaryotic translation initiation factor 2, subunit 3W89599−1.8 FBW5F-box and WD-40 domain protein 5W79991−2.1 PRSS20/KLK11HippostasinW13212−6.0 L7Ribosomal protein, mitochondrialAA028352−2.0 Rnasep2Ribonuclease P2W71337−1.7 Ube2g2Ubiquitin-conjugating enzyme E2G 2AA237600−11.2 Ube4Ubiquitin-conjugating enzyme 4AA108185−1.8Signal transduction ArhnAplysia Ras-related homolog N (RhoN)W64242−2.7 Bmp1Bone morphogenetic protein 1W82677−1.7 CD97CD97 antigenAA118715−1.8 Ddr1Receptor-like tyrosine kinaseW98395−5.8 Fem1bMouse feminization 1 b homolog (Caenorhabditis elegans)AA030303−14.1 ligpInterferon-inducible GTPaseAA260490+2.0 Map4k6Mitogen-activated protein kinase 6W64920−2.7Transcription Aebp2AE-binding protein 2AA416308−6.1 Atf1Activating transcription factor 1W87965−2.1 ErfEts2 repressor factorW70986−2.2 Esr1Estrogen receptor 1 (α)AA023625−1.6 Etv6ETS variant gene 6 (Tel oncogene)AA260520−5.5 Fkhr1Forkhead protein 1W36356−2.4 Hoxa2Homeobox A2W63822−2.6 Klf1Kruppel-like factor 1 (erythroid)W97446−1.7 NufipNuclear FMRP-interacting proteinAA139817−1.6 Pitx3Paired-like homeodomain transcription factor 3AA062140−3.9Transport facilitation BzrpBenzodiazepine receptor, peripheralW82946−4.2 Slc20a2Solute carrier family 20, member 2AA120631−2.5 P24AMouse cop-coated vesicle membrane protein p24 precursorW14393−1.6 Syt4Synaptotagmin 4W42224−2.6 Tor1aTorsin family 1, member AAA422787−10.5 TTRTransthyretinW17647−1.6The table shows a list of known genes, arranged into functional categories, that were found to be either up- or down-regulated in Pax6 heterozygous lenses. Open table in a new tab Table IIIList of known genes without a designated function that were abnormally expressed in Pax6 heterozygous lensesDescriptionGenBank™ accession no.Change-foldAcupuncture-induced gene 1AA014195−1.7Expressed sequence 2 embryonic lethal (Es2el)W34498−1.7LIM domains containing protein 1 (Limd1)AA204257−1.8Mouse zinc finger protein 261 (Zfp261)AA060697−2.6Mouse fatso (Fto)W33853−2.3Palmitoylated membrane protein p55AA003740−1.8Tal1 interrupting locusW75881−2.5Tangerin AAA217475−3.2Trefoil factor 1W83072−1.6 Open table in a new tab Table IVList of ESTs found to be abnormally expressed in Pax6 heterozygous lensesDescriptionGenBank™ accession no.Change-foldESTs, highly similar to α-actinin, smooth muscle isoform (Gallus gallus)W98655−2.2ESTs, highly similar to citrate synthase, mitochondrial precursor (Sus scrofa)W14146−2.5ESTs, highly similar to maternal pumilio protein (D. melanogaster)AA060266−2.2ESTs, highly similar to NADH-ubiquinone oxidoreductase B12 subunit (Bos taurus)W34455−2.2ESTs, highly similar to NEDD-4 protein (H. sapiens)AA118878−2.7ESTs, highly similar to neurolysin precursor (R. norvegicus)AA124221−3.4ESTs, highly similar to T-kininogen II precursor (R. norvegicus)W70823−2.9ESTs, highly similar to transcription factor IIIA (Xenopus laevis)W96919−2.6ESTs, highly similar to tyrosine-protein kinase JAK1 (H. sapien
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