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Expression of GATA6 in the human and mouse central nervous system
Developmental Brain Research 160 (2005) 90 – 95 www.elsevier.com/locate/devbrainres
Short Communication
Expression of GATA6 in the human and mouse central nervous system Deepak Kamnasaran a, Abhijit Guha a,b,* a
The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8 b Room 4W-446, Division of Neurosurgery, Toronto Western Hospital, 399 Bathurst St., Toronto, Ontario, Canada M5T 2S8 Accepted 29 July 2005 Available online 15 September 2005
Abstract The mammalian GATA family of transcription factors comprises of 6 members that are involved in diverse roles. The expression profile of GATA6 has been poorly defined in the central nervous system (CNS). In this report, we identify GATA6 expression in the normal mouse and human CNS, using Northern blot analyses, immunohistochemistry (IHC), and immunofluorescence (IF). GATA6 is expressed as a 2.2 kb transcript in the adult mouse brain and several regions of the adult human brain. Furthermore, cellular characterization demonstrates GATA6 nuclear expression in neurons, astrocytes, choroids plexus epithelium, and endothelial cells. D 2005 Elsevier B.V. All rights reserved. Theme: Gene expression Topic: Expression profiling in the central nervous system Keywords: Brain development; GATA6; Neurogenesis; Brain expression; Central nervous system
The mammalian GATA family of transcription factors, comprising of six members, contains two highly conserved zinc-finger DNA binding domains that interact with a canonical DNA motif (G/A)GATA(A/T) [10]. GATA-1/2/3 members are most frequently associated with the development of nervous system and hematopoietic cell lineages [3,12]. Whereas, GATA-4/5/6 are involved in the development of several organs such as the heart, gut, ovary, vascular, and extra-embryonic tissues [8,9,12]. We have focused on Gata6, which is expressed early in the blastocyst stage [8], as our ongoing work on gliomagenesis has identified Gata6 as a potential tumor suppressor gene. A homozygous null allele of the murine Gata6 gene was previously reported to result in embryonic lethality at E5.5 to E6.5, as a result of defects in endoderm differentiation
Abbreviations: CNS, central nervous system; IHC, immunohistochemistry; IF, immunofluorescence * Corresponding author. Room 4W-446, Division of Neurosurgery, Western Hospital, 399 Bathurst St., Toronto, Ontario, Canada M5T 2S8. Fax: +1 416 603 5298. E-mail address: [email protected] (A. Guha). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.07.012
[11]. The heterozygous mice were normal [11]. In the mouse, GATA6 has been discerned as a co-regulator with other transcription factors such as SF1 in the development of the adrenal gland [13], TTF1 in the development of the lung [7], and FOG in the development of the ovary [9]. Moreover, in the mouse, genes regulated by GATA6 include Wnt7b in the development of the lung [18], Hnf4 in the development of the viscera [11], Dab2 in the development of the ovary [4], and Plau in the development of vascular structures [14]. GATA6 is reported to be expressed in cranial neural crest cells and head mesenchyme [2]; however, knowledge on expression in the CNS is sparse. Using random mutagenesis by retroviral Gene Trapping, we identified GATA6 as a genetic modifier with oncogenic p21-Ras in a mouse model for astrocytoma (unpublished data). Our mouse model for astrocytoma was created using embryonic stem cell transgenesis to over-express oncogenic V12-Ha-Ras in astrocytes under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter [6]. One strain of our GFAP:V12-Ha-Ras transgenic mice (RasB8), developed low- and high-grade astrocytomas by 3 months of age, leading to death [6]. Primary astrocyte cultures established
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from the newborn pups did not transform or formed tumors in syngeneic Nod-Scid immunocompromised mice, in contrary to the astrocytes established from the 3 month RasB8 mice. GATA6 was identified as a tumor suppressor gene for astrocytomas in this mouse model, to accelerate proliferation, and subsequently transformation of newborn astrocytes, leading to anchorage independent growth in soft agarose, and the development of high-grade astrocytoma in syngeneic Nod-Scid mice (unpublished data). As part of our goals to decipher the functional role of GATA6 in gliomagenesis, we determined its geographic and cellular expression profile in normal adult human and mouse CNS. The human GATA6 gene, mapping to chromosome 18q11.1– q11.2, has seven exons spanning about 32.8 kb of genomic DNA. The 1st and part of the 2nd and 7th exons contain UTRs [1,17]. An upstream open reading frame (uORF), about eight amino acids in length, is proposed to control the translational stability of a long-type GATA6 protein [17]. Although expression of the human GATA6
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gene was reported as weak in brain [16], preliminary in silico expression profiling has demonstrated the isolation of an Expressed Sequence Tag (EST) from human brain (GeneCards, bioinformatics.weizmann.ac.il/cards). We cloned a partial 944 bp cDNA probe spanning the 7th exon by standard PCR and using a Marathon Ready adult human brain cDNA library (Clontech) as a template and the primer pairs (F-AAAGTGATTCTCGTGCCTTT, R-CAACCTGCCTGTGGGTTAGT). The PCR cycle was 94 -C for 3 min of denaturation, followed by 30 cycles of 94 -C – 30 s, 58 -C annealing, and 68 -C – 1 min extension. The cDNA probe (confirmed by sequencing) was labeled with 32P-a-dCTP using the RediPrimeII Kit (Amersham Pharmacia), then subjected to Northern analyses on Multi-tissue adult human brain blots II and V (Clontech) as specified by the manufacturer. The blots were exposed overnight to several days to obtain the most informative signals. GATA6 demonstrated ubiquitous expression in the human adult brain as a 2.2 kb transcript (Fig. 1A). Specifically, expression was noted in the
Fig. 1. GATA6 expression in adult human CNS. (A) Northern analysis with a human GATA6 partial cDNA probe. A ¨2.2 kb transcript is noted in several regions of the adult human brain using human adult brain MTN blots II and V (Clontech). Beta actin serves as a control. (B – D) Immunohistochemistry with GATA6-H92 primary polyclonal antibody (Santa Cruz) on normal human brain cortical specimens: (B) GATA6 immunoreactivity is detected in several subtypes of nuclei in the cortex of the frontal lobe; (C) GATA6 expression is noted in large neuronal nuclei (arrowhead) of the cortex of the frontal lobe; (D) GATA6 expression is noted in endothelial cells (*) of the cortex in the posterior fossa. (E) RT-PCR testing of GATA6 expression in normal human brain and immortalized normal human astrocytes. Templates used with the absence of Reverse transcriptase serve as a negative control. GADPH serves as a control. (F) Immunofluorescent localization of GATA6 expression in immortalized normal human astrocytes. Nuclear localization of GATA6 (top) is noted using Cy5conjugated GATA6-H92 polyclonal antibody (Santa Cruz). Cytoplasmic localization of GFAP (bottom) is noted using a Cy3-conjugated GFAP polyclonal antibody (Dako). The nucleus (center) is stained with DAPI (blue). (G) Immunofluorescent nuclear localization of GATA6 in the U343 MG cell line transiently transfected with 10 Ag/ml of Doxcycline inducible expression of murine GATA6. No GATA6 protein expression is noted with the untransfected U343 MG cell line (negative control). Nuclear localization of GATA6 is noted in the transiently transfected cells using Cy5-conjugated GATA6-H92 polyclonal antibody (Santa Cruz). The nucleus (center) is stained with DAPI (blue).
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amygdala, caudate nucleus, corpus callosum, hippocampus, thalamus, cerebellum, cerebral cortex, medulla, putamen, and the temporal, occipital, and frontal lobes. In order to determine the cellular expression profile of GATA6 in the CNS, immunohistochemistry (IHC) was undertaken on normal human brain operative specimens. Paraffin embedded normal human brain operative specimens were sectioned into 6 Am, then subjected to standard IHC protocols, with heat inactivation antigen retrieval (pressure cooking in Antigen unmasking solution (Vector Laboratories Inc.) for 15 min), and using 1 in 100 dilution of primary rabbit GATA6-H92 polyclonal antibody (Santa Cruz) after pre-blocking in serum for 4 days, 1:1000 dilution of biotinylated secondary Protein G antibody and detection with a Vectastain ABC-Universal kit (Vector Laboratories Inc.). GATA6 expression was noted in astrocytes, neurons, and endothelial cells surrounding blood vessels (Figs. 1B – D). Confirmation of GATA6 expression in astrocytes by RTPCR analysis was undertaken. Total RNA was extracted from normal human brain operative specimen and immor-
talized normal human astrocytes (with hTERT, a gift from Dr. R. Peiper (UCSF)), using the RNAeasy Mini Kit (Qiagen). The immortalized normal human astrocytes, which cannot transform by anchorage independent growth in soft agarose, or develop tumors in syngeneic immunocompromised mice, have been previously characterized and are abundant in GFAP immunopositivity (100%) [15]. First strand cDNA was made from 2 Ag of Total RNA using the One Step RT-PCR kit (Qiagen), and before being subjected to standard PCR with the GATA6 primer pair (F-TTCCCATGACTCCAACTTCC, R-CGCCTATGTAGAGCCCATCT), GAPDH control primer pair (F-TGCTGGAAAAATTGCAACAA, R-CAACCTGCCTGTGGGTTAGT) and PCR cycle (94 -C– 3 min of denaturation, and 30 cycles of 94 -C – 30 s, 58 -C – 30 s, 68 -C –1 s). Expression was noted in both normal brain and immortalized astrocytes (Fig. 1E). In order to confirm expression, we used IF as per standard protocols on the normal human immortalized astrocytes fixed with methanol and acetone after being grown overnight on 2-well chamber slides (Nalgene Nunc International). Rabbit GATA6 H92 polyclonal antibody (Santa
Fig. 2. GATA6 expression pattern in the mouse CNS. (A) Schematic of three Gata6 transcript isoforms. The black shaded boxes represent the open reading frame within the exons. (B) Determination of Gata6 transcript isoforms expressed in mouse adult heart and brain by PCR. An ¨1.8 kb Gata6 isoform 2 was discerned expressed (arrow) in adult brain, and verified by sequencing. 1.3 kb (isoform 1) and 3.7 kb (isoform 3) Gata6 transcript isoforms were noted expressed (arrow) in adult heart (positive control), and verified by sequencing. The other PCR products were determined as non-specific after sequence verification. No products were noted with negative control (replacement of template with water only). (C) Northern analysis with a murine GATA6 cDNA probe. A ¨2.2 kb transcript is noted in the adult brain using an adult mouse MTN blot (Clontech). Expression is also detected in other adult tissues such as the lung, liver, kidney, and testis. Beta actin serves as a control. (D) Immunohistochemistry with GATA6-H92 primary polyclonal antibody (Santa Cruz): GATA6 is diffusely expressed in the cortex of the frontal lobe in a newborn (1 week old) mouse brain. Absent expression is noted in the outer cortical layer.
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Cruz) was labeled with Cy5 and Rabbit Cow GFAP polyclonal antibody (Dako) was labeled with Cy3 using the Cy3/Cy5 Antibody labeling kit (Amersham Pharmacia). 1:500 dilution of Cy5-GATA6 (green) and 1:1000 dilution of Cy3-GFAP (red) were used in double immunofluorescence hybridizations. GATA6 expression was noted in the nucleus of human astrocytes (about 80%) (Fig. 1F). Partial expression is most likely due to the population of cells in heterogeneous cell cycle stages. To test the specificity of the Rabbit GATA6 H92 polycolonal antibody (Santa Cruz), the U343 MG cell line was transiently transfected with a plasmid encoding a Doxcycline inducible expression of murine Gata6 (pREV-Tre:Gata6-Beta-globin Poly A). The murine Gata6-Beta-globlin Poly A cassette (a gift from Dr. F Grosveld (Erasmus University Medical Centre, Rotterdam) was subcloned into the pREV-Tre vector (Clontech) prior to use. U343 MG cells lack GATA6 protein expression when untransfected, but transient transfected expression of GATA6 is noted upon induction with 10 Ag/ml of Doxycycline (Fig. 1G), implicating the specificity of the antibody. U343 MG is an established human malignant glioma cell line without any GATA6 mRNA or protein expression, characterized as part of our work on the role of GATA6 in gliomagenesis (unpublished results).
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The expression profile of GATA6 in the mouse CNS was also determined. The mouse GATA6 gene, mapping to chromosome 18A1, is postulated to contain three transcript isoforms: Isoform 1; Isoform 2; Isoform 3 (EMBL accession numbers: AF179425, NM_010258, AJ243146), that span a range of 2 – 7 exons (Fig. 2A). The genomic structure of one transcript (alternative 2, EMBL accession number NM_010258) is conserved with the human GATA6 with an estimated 70% identity [1]. As a preliminary test, standard PCR using Marathon Ready mouse adult brain and heart cDNA libraries (Clontech) as templates was undertaken, to determine which of the three alternative transcript isoforms is expressed. The following primer pairs were used: Isoform 1: F-GGTCTACGTGCCCACCAC, R-GCCAGAGCACACCAAGAATC; Isoform 2:F-GCCACCATGTACCAGACCCTCGCCG, R-TCAGGCCAGGGCCAGAQGCACACCA; Isoform 3:F-GCCACCATGGCCTTGACTGACGGC, RTCAGCCTCTGAGCCTGCCACGTAGAAAA, with the following PCR cycles: 94 -C – 3 min initial denaturation, followed by 50 cycles of 94 -C –30 s, 58 -C –30 s, 68 -C of extension, specifically 1 min for Isoform 1; 2 min for Isoform 2; 3 min for Isoform 3. Only Isoform 2 (EMBL accession number NM_010258) was discerned as expressed in adult mouse brain (Fig. 2B). Isoforms 1 and 3 (EMBL accession
Fig. 3. GATA6 expression in different cell types in the mouse CNS. Immunohistochemistry with a GATA6-H92 primary polyclonal antibody (Santa Cruz). (A) GATA6 expression is noted diffusely in the adult (2 months old) mouse cortex and hippocampus and in the nuclei of several cell types. GATA6 expression is noted in: (B) choroid plexus epithelial cells (arrows) close to the lateral ventricle, (C) endothelial cells (*) of the posterior hypothalamic area, (D) neurons (arrowhead) of the frontal cortex. (E) Immunofluorescent localization of GATA6 expression in immortalized normal murine astrocytes (with T-antigen). Nuclear localization of GATA6 (left) is noted using a Cy5-conjugated GATA6-H92 polyclonal antibody (Santa Cruz). Cytoplasmic localization of GFAP (right) is noted using a Cy3-conjugated GFAP polyclonal antibody (Dako). The nucleus (nucleus) is stained with DAPI (blue).
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numbers AF179425, AJ243146) were noted expressed in the adult heart (positive control) (Fig. 2B). Isoform 3 was previously cloned from the murine embryonic and adult heart, and whole embryo (E10.5) [1]. Isoform 3 was noted with expression in tissues such as the murine embryonic urogenital tract, heart, umbilical cord, and adult heart, but absent in expression in the head [1]. Isoform 3 may encode a transcript variant that regulates the other Gata6 transcript isoforms as demonstrated with the PITX2 gene [5]. The cDNA of transcript Isoform 2 (verified by sequencing) was labeled using the RediPrimeII Kit (Amersham Pharmacia) with 32P-a-dCTP and hybridized to a panel of Mouse adult multi-tissue Northern blot (Clontech). The blot was exposed overnight, with expression of a 2.2 kb transcript noted in the mouse adult brain (Fig. 2C). Furthermore, expression of other transcript isoforms was noted in the lung (1.7 kb), liver (4.2 kb), kidney (2.4 kb), and testis (2.2 kb) (Fig. 2C), as reported previously [16]. During the ages of 1 week, 1 month, and 2 months, Gata6 expression was determined in the CNS of the mouse. With the use of RNA in situ hybridization, Gata6 expression was previously noted in the developing prosencephalon, mesencephalon, rhombencephalon, and spinal column at E11, with more confined expression in the frontal lobe and posterior fossa at E15 and 1 week (Brain Expression Map Project, www.stjudebgem.org/web/mainPage/mainPage.php). Moreover, Gata6 expression during mid-gestation was reported in cranial neural crest cells and head mesenchyme [2]. To document GATA6 expression, IHC on 6 Am paraffin embedded mouse brain sections was undertaken with the Rabbit GATA6-H92 polyclonal antibody (Santa Cruz), using similar conditions as the normal human brain operative specimens. GATA6 expression was noted diffusely throughout the newborn (1 week) brain, but with some absent expression in the outer cortical layer of the frontal lobe (Fig. 2D). GATA6 expression is continually diffused in 1 and 2 month adult brains (Fig. 3A), but is most prominent in the cerebellum and ventricular linings. Our IHC temporal and spatial expression findings are in concordance with the previously reported RNA in situ findings of GATA6 expression (Brain Expression Map Project). Furthermore, GATA6 expression was prominent in neurons, endothelial cells, and choroid plexus epithelium (Figs. 3B – D). In order to confirm expression in astrocytes, we used IF as per standard protocols and conditions specified above on normal mouse immortalized astrocytes (with T-antigen). We have previously isolated primary murine astrocytes from a 1 week pup (unpublished data). Over 95% immunopositivity for the astrocyte marker GFAP was noted, and served as a criteria before subjecting the primary astrocyte culture to immortalization with T-antigen. GATA6 expression was noted in the nucleus of 40– 50% murine astrocytes (Fig. 3E). Similar to the immortalized normal human astrocytes, variable GATA6 expression may be a consequence of cells in heterogeneous cell cycle stages.
In summary, our work has demonstrated expression of GATA6 at the RNA and protein levels in the normal mouse and human adult CNS. We have identified 3 cell types (astrocytes, neurons, and endothelial cells) where GATA6 is expressed in the adult human brain and an RNA transcript isoform that is ubiquitously expressed. We have also identified one of three alternative transcript isforms that is expressed in the murine adult brain, with GATA6 expression spatio-temporally diffused during development, and in four cell types (astrocytes, neurons, endothelial cells, choroids plexus epithelium). Our ongoing research is highly suggestive that GATA6 is a tumor suppressor gene in human and mouse gliomagenesis (unpublished data). The findings reported herein are precursory for additional research to elucidate the role of GATA6 in the development and function of the CNS and how its loss may contribute to gliomagenesis.
Acknowledgments We thank Mr. Kevin So of the Clinical Research Program (CRP) (Toronto General Hospital) for assistance with the IHC experiments, Dr. Cynthia Hawkins (Hospital for Sick Children Research Institute) for histological assistance, Dr. Russ Pieper for providing us with immortalized normal human astrocytes (hTERT), and Dr. Frank Grosveld for providing us a plasmid with the murine Gata6 open reading frame. D.K. is a recipient of funds from Restracomp, Neurooncology Basic Research Fellowship, NSERC, and AHFMR. The project was funded by grants to A.G. from NCIC, Cleveland Foundation and NIH.
References [1] A. Brewer, C. Gove, A. Davies, C. McNulty, D. Barrow, M. Koutsourakis, F. Farzaneh, J. Pizzey, A. Bomford, R. Patient, The human and mouse GATA-6 genes utilize two promoters and two initiation codons, J. Biol. Chem. 274 (1999) 38004 – 38016. [2] A. Brewer, G. Nemer, C. Gove, F. Rawlins, M. Nemer, R. Patient, J. Pizzey, Widespread expression of an extended peptide sequence of GATA-6 during murine embryogenesis and non-equivalence of RNA and protein expression domains, Gene Expression Patterns 2 (2002) 123 – 131. [3] A.B. Cantor, S.H. Orkin, Transcriptional regulation of erythropoiesis: an affair involving multiple partners, Oncogene 21 (2002) 3368 – 3376. [4] C.D. Capo-chichi, I.H. Roland, L. Vanderveer, R. Bao, T. Yamagata, H. Hirai, C. Cohen, T.C. Hamilton, A.K. Godwin, X.X. Xu, Anomalous expression of epithelial differentiation-determining GATA factors in ovarian tumorigenesis, Cancer Res. 63 (2003) 4967 – 4977. [5] C.J. Cox, H.M. Espinoza, B. McWilliams, K. Chappell, L. Morton, T.A. Hjalt, E.V. Semina, B.A. Amendt, Differential regulation of gene expression by PITX2 isoforms, J. Biol. Chem. 277 (2002) 25001 – 25010. [6] H. Ding, L. Roncari, P. Shannon, X. Wu, N. Lau, J. Karaskova, D.H. Gutmann, J.A. Squire, A. Nagy, A. Guha, Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in
D. Kamnasaran, A. Guha / Developmental Brain Research 160 (2005) 90 – 95
[7]
[8]
[9]
[10]
[11]
[12]
a transgenic mouse model of human gliomas, Cancer Res. 61 (2001) 3826 – 3836. R. Keijzer, M. van Tuyl, C. Meijers, M. Post, D. Tibboel, F. Grosveld, M. Koutsourakis, The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development, Development 128 (2001) 503 – 511. M. Koutsourakis, A. Langeveld, R. Patient, R. Beddington, F. Grosveld, The transcription factor GATA6 is essential for early extraembryonic development, Development 126 (1999) 723 – 732. M.P. Laitinen, M. Anttonen, I. Ketola, D.B. Wilson, O. Ritvos, R. Butzow, M. Heikinheimo, Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors, J. Clin. Endocrinol. Metab. 85 (9) (2000 Sep.) 3476 – 3483. J.A. Lowry, W.R. Atchley, Molecular evolution of the GATA family of transcription factors: conservation within the DNA-binding domain, J. Mol. Evol. 50 (2000) 103 – 115. E.E. Morrisey, Z. Tang, K. Sigrist, M.M. Lu, F. Jiang, H.S. Ip, M.S. Parmacek, GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo, Genes Dev. 12 (1998) 3579 – 3590. R.K. Patient, J.D. McGhee, The GATA family (vertebrates and invertebrates), Curr. Opin. Genet. Dev. 12 (2002) 416 – 422.
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[13] K.J. Saner, T. Suzuki, H. Sasano, J. Pizzey, C. Ho, J.F. Strauss III, B.R. Carr, W.E. Rainey, Steroid sulfotransferase 2A1 gene transcription is regulated by steroidogenic factor 1 and GATA-6 in the human adrenal, Mol. Endocrinol. 19 (2005) 184 – 197. [14] T. Sokabe, K. Yamamoto, N. Ohura, H. Nakatsuka, K. Qin, S. Obi, A. Kamiya, J. Ando, Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells, Am. J. Physiol.: Heart Circ. Physiol. 287 (2004) H2027 – H2034. [15] Y. Sonoda, T. Ozawa, Y. Hirose, K.D. Aldape, M. McMahon, M.S. Berger, R.O. Pieper, Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma, Cancer Res. 61 (2001) 4956 – 4960. [16] E. Suzuki, T. Evans, J. Lowry, L. Truong, D.W. Bell, J.R. Testa, K. Walsh, The human GATA-6 gene: structure, chromosomal location, and regulation of expression by tissue-specific and mitogen-responsive signals, Genomics 38 (1996) 283 – 290. [17] M. Takeda, K. Obayashi, A. Kobayashi, M. Maeda, A unique role of an amino terminal 16-residue region of long-type GATA-6, J. Biochem. (Tokyo) 135 (2004) 639 – 650. [18] J. Weidenfeld, W. Shu, L. Zhang, S.E. Millar, E.E. Morrisey, The WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in lung epithelium, J. Biol. Chem. 277 (2002) 21061 – 21070.
Short Communication
Expression of GATA6 in the human and mouse central nervous system Deepak Kamnasaran a, Abhijit Guha a,b,* a
The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8 b Room 4W-446, Division of Neurosurgery, Toronto Western Hospital, 399 Bathurst St., Toronto, Ontario, Canada M5T 2S8 Accepted 29 July 2005 Available online 15 September 2005
Abstract The mammalian GATA family of transcription factors comprises of 6 members that are involved in diverse roles. The expression profile of GATA6 has been poorly defined in the central nervous system (CNS). In this report, we identify GATA6 expression in the normal mouse and human CNS, using Northern blot analyses, immunohistochemistry (IHC), and immunofluorescence (IF). GATA6 is expressed as a 2.2 kb transcript in the adult mouse brain and several regions of the adult human brain. Furthermore, cellular characterization demonstrates GATA6 nuclear expression in neurons, astrocytes, choroids plexus epithelium, and endothelial cells. D 2005 Elsevier B.V. All rights reserved. Theme: Gene expression Topic: Expression profiling in the central nervous system Keywords: Brain development; GATA6; Neurogenesis; Brain expression; Central nervous system
The mammalian GATA family of transcription factors, comprising of six members, contains two highly conserved zinc-finger DNA binding domains that interact with a canonical DNA motif (G/A)GATA(A/T) [10]. GATA-1/2/3 members are most frequently associated with the development of nervous system and hematopoietic cell lineages [3,12]. Whereas, GATA-4/5/6 are involved in the development of several organs such as the heart, gut, ovary, vascular, and extra-embryonic tissues [8,9,12]. We have focused on Gata6, which is expressed early in the blastocyst stage [8], as our ongoing work on gliomagenesis has identified Gata6 as a potential tumor suppressor gene. A homozygous null allele of the murine Gata6 gene was previously reported to result in embryonic lethality at E5.5 to E6.5, as a result of defects in endoderm differentiation
Abbreviations: CNS, central nervous system; IHC, immunohistochemistry; IF, immunofluorescence * Corresponding author. Room 4W-446, Division of Neurosurgery, Western Hospital, 399 Bathurst St., Toronto, Ontario, Canada M5T 2S8. Fax: +1 416 603 5298. E-mail address: [email protected] (A. Guha). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.07.012
[11]. The heterozygous mice were normal [11]. In the mouse, GATA6 has been discerned as a co-regulator with other transcription factors such as SF1 in the development of the adrenal gland [13], TTF1 in the development of the lung [7], and FOG in the development of the ovary [9]. Moreover, in the mouse, genes regulated by GATA6 include Wnt7b in the development of the lung [18], Hnf4 in the development of the viscera [11], Dab2 in the development of the ovary [4], and Plau in the development of vascular structures [14]. GATA6 is reported to be expressed in cranial neural crest cells and head mesenchyme [2]; however, knowledge on expression in the CNS is sparse. Using random mutagenesis by retroviral Gene Trapping, we identified GATA6 as a genetic modifier with oncogenic p21-Ras in a mouse model for astrocytoma (unpublished data). Our mouse model for astrocytoma was created using embryonic stem cell transgenesis to over-express oncogenic V12-Ha-Ras in astrocytes under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter [6]. One strain of our GFAP:V12-Ha-Ras transgenic mice (RasB8), developed low- and high-grade astrocytomas by 3 months of age, leading to death [6]. Primary astrocyte cultures established
D. Kamnasaran, A. Guha / Developmental Brain Research 160 (2005) 90 – 95
from the newborn pups did not transform or formed tumors in syngeneic Nod-Scid immunocompromised mice, in contrary to the astrocytes established from the 3 month RasB8 mice. GATA6 was identified as a tumor suppressor gene for astrocytomas in this mouse model, to accelerate proliferation, and subsequently transformation of newborn astrocytes, leading to anchorage independent growth in soft agarose, and the development of high-grade astrocytoma in syngeneic Nod-Scid mice (unpublished data). As part of our goals to decipher the functional role of GATA6 in gliomagenesis, we determined its geographic and cellular expression profile in normal adult human and mouse CNS. The human GATA6 gene, mapping to chromosome 18q11.1– q11.2, has seven exons spanning about 32.8 kb of genomic DNA. The 1st and part of the 2nd and 7th exons contain UTRs [1,17]. An upstream open reading frame (uORF), about eight amino acids in length, is proposed to control the translational stability of a long-type GATA6 protein [17]. Although expression of the human GATA6
91
gene was reported as weak in brain [16], preliminary in silico expression profiling has demonstrated the isolation of an Expressed Sequence Tag (EST) from human brain (GeneCards, bioinformatics.weizmann.ac.il/cards). We cloned a partial 944 bp cDNA probe spanning the 7th exon by standard PCR and using a Marathon Ready adult human brain cDNA library (Clontech) as a template and the primer pairs (F-AAAGTGATTCTCGTGCCTTT, R-CAACCTGCCTGTGGGTTAGT). The PCR cycle was 94 -C for 3 min of denaturation, followed by 30 cycles of 94 -C – 30 s, 58 -C annealing, and 68 -C – 1 min extension. The cDNA probe (confirmed by sequencing) was labeled with 32P-a-dCTP using the RediPrimeII Kit (Amersham Pharmacia), then subjected to Northern analyses on Multi-tissue adult human brain blots II and V (Clontech) as specified by the manufacturer. The blots were exposed overnight to several days to obtain the most informative signals. GATA6 demonstrated ubiquitous expression in the human adult brain as a 2.2 kb transcript (Fig. 1A). Specifically, expression was noted in the
Fig. 1. GATA6 expression in adult human CNS. (A) Northern analysis with a human GATA6 partial cDNA probe. A ¨2.2 kb transcript is noted in several regions of the adult human brain using human adult brain MTN blots II and V (Clontech). Beta actin serves as a control. (B – D) Immunohistochemistry with GATA6-H92 primary polyclonal antibody (Santa Cruz) on normal human brain cortical specimens: (B) GATA6 immunoreactivity is detected in several subtypes of nuclei in the cortex of the frontal lobe; (C) GATA6 expression is noted in large neuronal nuclei (arrowhead) of the cortex of the frontal lobe; (D) GATA6 expression is noted in endothelial cells (*) of the cortex in the posterior fossa. (E) RT-PCR testing of GATA6 expression in normal human brain and immortalized normal human astrocytes. Templates used with the absence of Reverse transcriptase serve as a negative control. GADPH serves as a control. (F) Immunofluorescent localization of GATA6 expression in immortalized normal human astrocytes. Nuclear localization of GATA6 (top) is noted using Cy5conjugated GATA6-H92 polyclonal antibody (Santa Cruz). Cytoplasmic localization of GFAP (bottom) is noted using a Cy3-conjugated GFAP polyclonal antibody (Dako). The nucleus (center) is stained with DAPI (blue). (G) Immunofluorescent nuclear localization of GATA6 in the U343 MG cell line transiently transfected with 10 Ag/ml of Doxcycline inducible expression of murine GATA6. No GATA6 protein expression is noted with the untransfected U343 MG cell line (negative control). Nuclear localization of GATA6 is noted in the transiently transfected cells using Cy5-conjugated GATA6-H92 polyclonal antibody (Santa Cruz). The nucleus (center) is stained with DAPI (blue).
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amygdala, caudate nucleus, corpus callosum, hippocampus, thalamus, cerebellum, cerebral cortex, medulla, putamen, and the temporal, occipital, and frontal lobes. In order to determine the cellular expression profile of GATA6 in the CNS, immunohistochemistry (IHC) was undertaken on normal human brain operative specimens. Paraffin embedded normal human brain operative specimens were sectioned into 6 Am, then subjected to standard IHC protocols, with heat inactivation antigen retrieval (pressure cooking in Antigen unmasking solution (Vector Laboratories Inc.) for 15 min), and using 1 in 100 dilution of primary rabbit GATA6-H92 polyclonal antibody (Santa Cruz) after pre-blocking in serum for 4 days, 1:1000 dilution of biotinylated secondary Protein G antibody and detection with a Vectastain ABC-Universal kit (Vector Laboratories Inc.). GATA6 expression was noted in astrocytes, neurons, and endothelial cells surrounding blood vessels (Figs. 1B – D). Confirmation of GATA6 expression in astrocytes by RTPCR analysis was undertaken. Total RNA was extracted from normal human brain operative specimen and immor-
talized normal human astrocytes (with hTERT, a gift from Dr. R. Peiper (UCSF)), using the RNAeasy Mini Kit (Qiagen). The immortalized normal human astrocytes, which cannot transform by anchorage independent growth in soft agarose, or develop tumors in syngeneic immunocompromised mice, have been previously characterized and are abundant in GFAP immunopositivity (100%) [15]. First strand cDNA was made from 2 Ag of Total RNA using the One Step RT-PCR kit (Qiagen), and before being subjected to standard PCR with the GATA6 primer pair (F-TTCCCATGACTCCAACTTCC, R-CGCCTATGTAGAGCCCATCT), GAPDH control primer pair (F-TGCTGGAAAAATTGCAACAA, R-CAACCTGCCTGTGGGTTAGT) and PCR cycle (94 -C– 3 min of denaturation, and 30 cycles of 94 -C – 30 s, 58 -C – 30 s, 68 -C –1 s). Expression was noted in both normal brain and immortalized astrocytes (Fig. 1E). In order to confirm expression, we used IF as per standard protocols on the normal human immortalized astrocytes fixed with methanol and acetone after being grown overnight on 2-well chamber slides (Nalgene Nunc International). Rabbit GATA6 H92 polyclonal antibody (Santa
Fig. 2. GATA6 expression pattern in the mouse CNS. (A) Schematic of three Gata6 transcript isoforms. The black shaded boxes represent the open reading frame within the exons. (B) Determination of Gata6 transcript isoforms expressed in mouse adult heart and brain by PCR. An ¨1.8 kb Gata6 isoform 2 was discerned expressed (arrow) in adult brain, and verified by sequencing. 1.3 kb (isoform 1) and 3.7 kb (isoform 3) Gata6 transcript isoforms were noted expressed (arrow) in adult heart (positive control), and verified by sequencing. The other PCR products were determined as non-specific after sequence verification. No products were noted with negative control (replacement of template with water only). (C) Northern analysis with a murine GATA6 cDNA probe. A ¨2.2 kb transcript is noted in the adult brain using an adult mouse MTN blot (Clontech). Expression is also detected in other adult tissues such as the lung, liver, kidney, and testis. Beta actin serves as a control. (D) Immunohistochemistry with GATA6-H92 primary polyclonal antibody (Santa Cruz): GATA6 is diffusely expressed in the cortex of the frontal lobe in a newborn (1 week old) mouse brain. Absent expression is noted in the outer cortical layer.
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Cruz) was labeled with Cy5 and Rabbit Cow GFAP polyclonal antibody (Dako) was labeled with Cy3 using the Cy3/Cy5 Antibody labeling kit (Amersham Pharmacia). 1:500 dilution of Cy5-GATA6 (green) and 1:1000 dilution of Cy3-GFAP (red) were used in double immunofluorescence hybridizations. GATA6 expression was noted in the nucleus of human astrocytes (about 80%) (Fig. 1F). Partial expression is most likely due to the population of cells in heterogeneous cell cycle stages. To test the specificity of the Rabbit GATA6 H92 polycolonal antibody (Santa Cruz), the U343 MG cell line was transiently transfected with a plasmid encoding a Doxcycline inducible expression of murine Gata6 (pREV-Tre:Gata6-Beta-globin Poly A). The murine Gata6-Beta-globlin Poly A cassette (a gift from Dr. F Grosveld (Erasmus University Medical Centre, Rotterdam) was subcloned into the pREV-Tre vector (Clontech) prior to use. U343 MG cells lack GATA6 protein expression when untransfected, but transient transfected expression of GATA6 is noted upon induction with 10 Ag/ml of Doxycycline (Fig. 1G), implicating the specificity of the antibody. U343 MG is an established human malignant glioma cell line without any GATA6 mRNA or protein expression, characterized as part of our work on the role of GATA6 in gliomagenesis (unpublished results).
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The expression profile of GATA6 in the mouse CNS was also determined. The mouse GATA6 gene, mapping to chromosome 18A1, is postulated to contain three transcript isoforms: Isoform 1; Isoform 2; Isoform 3 (EMBL accession numbers: AF179425, NM_010258, AJ243146), that span a range of 2 – 7 exons (Fig. 2A). The genomic structure of one transcript (alternative 2, EMBL accession number NM_010258) is conserved with the human GATA6 with an estimated 70% identity [1]. As a preliminary test, standard PCR using Marathon Ready mouse adult brain and heart cDNA libraries (Clontech) as templates was undertaken, to determine which of the three alternative transcript isoforms is expressed. The following primer pairs were used: Isoform 1: F-GGTCTACGTGCCCACCAC, R-GCCAGAGCACACCAAGAATC; Isoform 2:F-GCCACCATGTACCAGACCCTCGCCG, R-TCAGGCCAGGGCCAGAQGCACACCA; Isoform 3:F-GCCACCATGGCCTTGACTGACGGC, RTCAGCCTCTGAGCCTGCCACGTAGAAAA, with the following PCR cycles: 94 -C – 3 min initial denaturation, followed by 50 cycles of 94 -C –30 s, 58 -C –30 s, 68 -C of extension, specifically 1 min for Isoform 1; 2 min for Isoform 2; 3 min for Isoform 3. Only Isoform 2 (EMBL accession number NM_010258) was discerned as expressed in adult mouse brain (Fig. 2B). Isoforms 1 and 3 (EMBL accession
Fig. 3. GATA6 expression in different cell types in the mouse CNS. Immunohistochemistry with a GATA6-H92 primary polyclonal antibody (Santa Cruz). (A) GATA6 expression is noted diffusely in the adult (2 months old) mouse cortex and hippocampus and in the nuclei of several cell types. GATA6 expression is noted in: (B) choroid plexus epithelial cells (arrows) close to the lateral ventricle, (C) endothelial cells (*) of the posterior hypothalamic area, (D) neurons (arrowhead) of the frontal cortex. (E) Immunofluorescent localization of GATA6 expression in immortalized normal murine astrocytes (with T-antigen). Nuclear localization of GATA6 (left) is noted using a Cy5-conjugated GATA6-H92 polyclonal antibody (Santa Cruz). Cytoplasmic localization of GFAP (right) is noted using a Cy3-conjugated GFAP polyclonal antibody (Dako). The nucleus (nucleus) is stained with DAPI (blue).
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numbers AF179425, AJ243146) were noted expressed in the adult heart (positive control) (Fig. 2B). Isoform 3 was previously cloned from the murine embryonic and adult heart, and whole embryo (E10.5) [1]. Isoform 3 was noted with expression in tissues such as the murine embryonic urogenital tract, heart, umbilical cord, and adult heart, but absent in expression in the head [1]. Isoform 3 may encode a transcript variant that regulates the other Gata6 transcript isoforms as demonstrated with the PITX2 gene [5]. The cDNA of transcript Isoform 2 (verified by sequencing) was labeled using the RediPrimeII Kit (Amersham Pharmacia) with 32P-a-dCTP and hybridized to a panel of Mouse adult multi-tissue Northern blot (Clontech). The blot was exposed overnight, with expression of a 2.2 kb transcript noted in the mouse adult brain (Fig. 2C). Furthermore, expression of other transcript isoforms was noted in the lung (1.7 kb), liver (4.2 kb), kidney (2.4 kb), and testis (2.2 kb) (Fig. 2C), as reported previously [16]. During the ages of 1 week, 1 month, and 2 months, Gata6 expression was determined in the CNS of the mouse. With the use of RNA in situ hybridization, Gata6 expression was previously noted in the developing prosencephalon, mesencephalon, rhombencephalon, and spinal column at E11, with more confined expression in the frontal lobe and posterior fossa at E15 and 1 week (Brain Expression Map Project, www.stjudebgem.org/web/mainPage/mainPage.php). Moreover, Gata6 expression during mid-gestation was reported in cranial neural crest cells and head mesenchyme [2]. To document GATA6 expression, IHC on 6 Am paraffin embedded mouse brain sections was undertaken with the Rabbit GATA6-H92 polyclonal antibody (Santa Cruz), using similar conditions as the normal human brain operative specimens. GATA6 expression was noted diffusely throughout the newborn (1 week) brain, but with some absent expression in the outer cortical layer of the frontal lobe (Fig. 2D). GATA6 expression is continually diffused in 1 and 2 month adult brains (Fig. 3A), but is most prominent in the cerebellum and ventricular linings. Our IHC temporal and spatial expression findings are in concordance with the previously reported RNA in situ findings of GATA6 expression (Brain Expression Map Project). Furthermore, GATA6 expression was prominent in neurons, endothelial cells, and choroid plexus epithelium (Figs. 3B – D). In order to confirm expression in astrocytes, we used IF as per standard protocols and conditions specified above on normal mouse immortalized astrocytes (with T-antigen). We have previously isolated primary murine astrocytes from a 1 week pup (unpublished data). Over 95% immunopositivity for the astrocyte marker GFAP was noted, and served as a criteria before subjecting the primary astrocyte culture to immortalization with T-antigen. GATA6 expression was noted in the nucleus of 40– 50% murine astrocytes (Fig. 3E). Similar to the immortalized normal human astrocytes, variable GATA6 expression may be a consequence of cells in heterogeneous cell cycle stages.
In summary, our work has demonstrated expression of GATA6 at the RNA and protein levels in the normal mouse and human adult CNS. We have identified 3 cell types (astrocytes, neurons, and endothelial cells) where GATA6 is expressed in the adult human brain and an RNA transcript isoform that is ubiquitously expressed. We have also identified one of three alternative transcript isforms that is expressed in the murine adult brain, with GATA6 expression spatio-temporally diffused during development, and in four cell types (astrocytes, neurons, endothelial cells, choroids plexus epithelium). Our ongoing research is highly suggestive that GATA6 is a tumor suppressor gene in human and mouse gliomagenesis (unpublished data). The findings reported herein are precursory for additional research to elucidate the role of GATA6 in the development and function of the CNS and how its loss may contribute to gliomagenesis.
Acknowledgments We thank Mr. Kevin So of the Clinical Research Program (CRP) (Toronto General Hospital) for assistance with the IHC experiments, Dr. Cynthia Hawkins (Hospital for Sick Children Research Institute) for histological assistance, Dr. Russ Pieper for providing us with immortalized normal human astrocytes (hTERT), and Dr. Frank Grosveld for providing us a plasmid with the murine Gata6 open reading frame. D.K. is a recipient of funds from Restracomp, Neurooncology Basic Research Fellowship, NSERC, and AHFMR. The project was funded by grants to A.G. from NCIC, Cleveland Foundation and NIH.
References [1] A. Brewer, C. Gove, A. Davies, C. McNulty, D. Barrow, M. Koutsourakis, F. Farzaneh, J. Pizzey, A. Bomford, R. Patient, The human and mouse GATA-6 genes utilize two promoters and two initiation codons, J. Biol. Chem. 274 (1999) 38004 – 38016. [2] A. Brewer, G. Nemer, C. Gove, F. Rawlins, M. Nemer, R. Patient, J. Pizzey, Widespread expression of an extended peptide sequence of GATA-6 during murine embryogenesis and non-equivalence of RNA and protein expression domains, Gene Expression Patterns 2 (2002) 123 – 131. [3] A.B. Cantor, S.H. Orkin, Transcriptional regulation of erythropoiesis: an affair involving multiple partners, Oncogene 21 (2002) 3368 – 3376. [4] C.D. Capo-chichi, I.H. Roland, L. Vanderveer, R. Bao, T. Yamagata, H. Hirai, C. Cohen, T.C. Hamilton, A.K. Godwin, X.X. Xu, Anomalous expression of epithelial differentiation-determining GATA factors in ovarian tumorigenesis, Cancer Res. 63 (2003) 4967 – 4977. [5] C.J. Cox, H.M. Espinoza, B. McWilliams, K. Chappell, L. Morton, T.A. Hjalt, E.V. Semina, B.A. Amendt, Differential regulation of gene expression by PITX2 isoforms, J. Biol. Chem. 277 (2002) 25001 – 25010. [6] H. Ding, L. Roncari, P. Shannon, X. Wu, N. Lau, J. Karaskova, D.H. Gutmann, J.A. Squire, A. Nagy, A. Guha, Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in
D. Kamnasaran, A. Guha / Developmental Brain Research 160 (2005) 90 – 95
[7]
[8]
[9]
[10]
[11]
[12]
a transgenic mouse model of human gliomas, Cancer Res. 61 (2001) 3826 – 3836. R. Keijzer, M. van Tuyl, C. Meijers, M. Post, D. Tibboel, F. Grosveld, M. Koutsourakis, The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development, Development 128 (2001) 503 – 511. M. Koutsourakis, A. Langeveld, R. Patient, R. Beddington, F. Grosveld, The transcription factor GATA6 is essential for early extraembryonic development, Development 126 (1999) 723 – 732. M.P. Laitinen, M. Anttonen, I. Ketola, D.B. Wilson, O. Ritvos, R. Butzow, M. Heikinheimo, Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors, J. Clin. Endocrinol. Metab. 85 (9) (2000 Sep.) 3476 – 3483. J.A. Lowry, W.R. Atchley, Molecular evolution of the GATA family of transcription factors: conservation within the DNA-binding domain, J. Mol. Evol. 50 (2000) 103 – 115. E.E. Morrisey, Z. Tang, K. Sigrist, M.M. Lu, F. Jiang, H.S. Ip, M.S. Parmacek, GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo, Genes Dev. 12 (1998) 3579 – 3590. R.K. Patient, J.D. McGhee, The GATA family (vertebrates and invertebrates), Curr. Opin. Genet. Dev. 12 (2002) 416 – 422.
95
[13] K.J. Saner, T. Suzuki, H. Sasano, J. Pizzey, C. Ho, J.F. Strauss III, B.R. Carr, W.E. Rainey, Steroid sulfotransferase 2A1 gene transcription is regulated by steroidogenic factor 1 and GATA-6 in the human adrenal, Mol. Endocrinol. 19 (2005) 184 – 197. [14] T. Sokabe, K. Yamamoto, N. Ohura, H. Nakatsuka, K. Qin, S. Obi, A. Kamiya, J. Ando, Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells, Am. J. Physiol.: Heart Circ. Physiol. 287 (2004) H2027 – H2034. [15] Y. Sonoda, T. Ozawa, Y. Hirose, K.D. Aldape, M. McMahon, M.S. Berger, R.O. Pieper, Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma, Cancer Res. 61 (2001) 4956 – 4960. [16] E. Suzuki, T. Evans, J. Lowry, L. Truong, D.W. Bell, J.R. Testa, K. Walsh, The human GATA-6 gene: structure, chromosomal location, and regulation of expression by tissue-specific and mitogen-responsive signals, Genomics 38 (1996) 283 – 290. [17] M. Takeda, K. Obayashi, A. Kobayashi, M. Maeda, A unique role of an amino terminal 16-residue region of long-type GATA-6, J. Biochem. (Tokyo) 135 (2004) 639 – 650. [18] J. Weidenfeld, W. Shu, L. Zhang, S.E. Millar, E.E. Morrisey, The WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in lung epithelium, J. Biol. Chem. 277 (2002) 21061 – 21070.