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1 Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
2 Department of Molecular and Developmental Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
3 Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan
4 Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA
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Introduction |
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The vertebrate GATA factor family consists of six members of zinc-finger transcription factors that usually recognize an (A/T)GATA(A/G) motif in GATA target genes (Yamamoto et al. 1990; Ohneda & Yamamoto 2002). Each GATA factor has a different expression profile, and each has been shown to play important roles in cell-type specific gene expression (Burch 2005). Several lines of recent evidence exploiting genetically engineered mice revealed that there exist multiple GATA-binding motifs in the enhancers of the genes encoding GATA1, GATA2, GATA5 and GATA6, and each of these motifs facilitate the autoregulation of GATA gene expression (Burch 2005). For example, GATA1 and GATA2 bind at different times to regulatory domains of the Gata1 gene to control its hematopoietic expression (reviewed in Ohneda & Yamamoto 2002). Similarly, we also identified an enhancer for the Gata2 gene that regulates its expression in early hematopoietic cells by recruiting GATA2 and/or GATA3 (Kobayashi-Osaki et al. 2005).
In order to fully elucidate the molecular basis of Gata2 gene expression, we have been analyzing the regulation of Gata2. These studies have revealed GATA2 contributions to cell-type specific gene expression and some of the regulatory domains within the gene that control GATA2 function in multiple tissues (Zhou et al. 2000; Khandekar et al. 2004, 2007; Kobayashi-Osaki et al. 2005). Importantly, we recently found that the regulatory motifs controlling the transcription of Gata2 are dispersed over approximately 413 kbp, even though the six coding exons of Gata2 span only 7 kbp (Brandt et al. 2008). The Gata2 gene harbors two separate urogenital enhancers at approximately +75 and +113 kbp from the translational initiation site, both of which are necessary for urogenital development (Khandekar et al. 2004). Hematopoietic enhancers have also been identified in the promoter proximal region of the gene (Kobayashi-Osaki et al. 2005; Grass et al. 2006). In addition to these upstream and downstream enhancers, we have also identified enhancers in introns 4 and 5 that dictate endothelial and spinal cord-specific expression of Gata2, respectively (Zhou et al. 2000; Khandekar et al. 2007).
We recently succeeded in visualization of Gata2 expression in a large battery of tissues where it is normally expressed by means of a green fluorescent protein (GFP) knock-in approach (Suzuki et al. 2006). The data showed that GATA2-expressing cells are widely distributed in neurons in the brain, spinal cord and sensory organs. However, the neural enhancer identified in the Gata2 gene intron 5 supports gene expression specifically in V2 interneurons of the spinal cord (Zhou et al. 2000). Therefore, we hypothesized that there should be additional enhancers that regulate Gata2 expression in the central nervous system (CNS). Indeed, our previous transgenic assays suggested that at least one additional brain enhancer might be located upstream of the Gata2 gene (Zhou et al. 2000).
To identify the putative Gata2 brain enhancer(s), we employed a standard transgenic mouse strategy. We found that two separate regulatory domains (referred to as the distal-5H and proximal-2H domains) located upstream of the transcriptional start site are both used by midbrain neural cells and hematopoietic cells. Interestingly, the enhancer usage differs between the two anatomically distinct midbrain sub-regions, the superior colliculus (SC) and inferior colliculus (IC). Both enhancers have the ability to independently activate Gata2 transcription in SC, but the expression in IC depends exclusively on the distal-5H domain. GATA2 activity was also found to be required for enhancer function in the midbrain, indicating that Gata2 gene expression is positively autoregulated. These results, taken together, demonstrate that the Gata2 gene expression in CNS is supported by cell-type specific enhancers acting through positive autoregulatory machinery.
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Results |
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To begin to address the mechanism(s) underlying Gata2 gene expression in the brain, we first examined reporter gene expression in a GFP knock-in ‘EII-KI’ mouse line (Gata2GFP/+ genotype). In this line, GFP was targeted to the Gata2 locus by homologous recombination (Suzuki et al. 2006), in order to monitor endogenous Gata2 gene expression by GFP. In the mouse brain, GFP expression was first observed in the midbrain at embryonic day 9.5 (E9.5; data not shown), and this expression was maintained at a high level throughout embryogenesis, as at E11.5 (Fig. 1A) and E15.0 (Fig. 1B), after birth (Fig. 1C) and even into the adult stage (data not shown). We also conducted reverse transcriptase-PCR (RT-PCR) analyses, and consistent with the GFP reporter study outlined above, the RT-PCR analysis confirmed that the endogenous Gata2 gene was exclusively expressed in the midbrain, but not in the cerebrum or cerebellum of the adult brain (Fig. 1D).
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GATA3 has also been reported to be expressed in the midbrain (Lakshmanan et al. 1999; Nardelli et al. 1999; van Doorninck et al. 1999) and the current RT-PCR analysis supports this conclusion. As is GATA2, GATA3 appears to be expressed in the midbrain in a tissue-restricted manner (Fig. 1D). To further examine GATA3 expression, we exploited a Gata3 gene-modified mouse in which the bacterial LacZ gene was knocked into the Gata3 locus (G3-KI or Gata3LacZ/+; Hendriks et al. 1999). We used the G3-KI mice for visualization of Gata3 expression during the embryonic stages, and found that the LacZ expression pattern was quite similar to that of the GFP reporter from the Gata2 locus (Fig. 1E). In E11.5 embryos, both GATA2 and GATA3 were expressed in the mesencephalon, metencephalon, otic vesicle and eye, but not in the diencephalon or telencephalon (Fig. 1A,E).
As the GFP and LacZ reporter analyses demonstrated concomitant expression of GATA2 and GATA3 in the midbrain, we next addressed whether GATA2 and GATA3 are co-expressed in the same cells or not. For this purpose, we generated double knock-in mutant mice (i.e. EII-KI:G3-KI) and analyzed Gata2-GFP and Gata3-LacZ expression in the same sections. As a result, we found that both Gata2 (GFP) and Gata3 (LacZ) were expressed in the superior colliculus (SC) and inferior colliculus (IC) of the midbrain at E11.5. Importantly, however, the GATA2 and GATA3 expression profiles were found to be mutually exclusive; a majority of midbrain cells expressed either GFP or LacZ, but not both (Fig. 1F). The Gata2 (GFP)-positive cells (brown in Fig. 1F) were mainly distributed in the inner and outer layers of the mesencephalon, surrounding the Gata3 (LacZ)-positive middle layer (blue). We also detected Gata2-GFP expression in brain vascular endothelial cells (identified by the ‘v’ in Fig. 1F), confirming our previous observation that endothelial cells expressing Gata2-GFP are widely distributed in the mouse (Kobayashi-Osaki et al. 2005; Khandekar et al. 2007). These results thus demonstrate that although a subset of midbrain cells express GATA2, a different set express GATA3 (Fig. 1F and Supplementary Fig. S1), suggesting that these two GATA factors play different roles in the specification of cellular identity in the mesencephalon. This is in contrast to the observation that GATA2 and GATA3 are co-expressed in the serotonergic neurons of the chick hindbrain (Craven et al. 2004).
As a preceding report indicated that GATA2 was expressed in serotonergic neurons (Craven et al. 2004), we next examined serotonin production in the midbrain GFP-positive cells by the means of immunohistochemistry using an anti-serotonin antibody. Indeed, there were a number of serotonin or GFP single positive cells in the dorsal raphe nucleus (DRN), and some cells were positive for both serotonin and GFP (Fig. 1M–O). However, the fact that a majority of GFP-positive cells are negative for serotonin production suggests that GATA2 may not play important roles for serotonin synthesis in the DRN. We failed to identify serotonin/GFP double positive cells in the SC or IC sub-regions of the adult mouse midbrain (Fig. 1G–L, respectively). These observations demonstrate that GATA2 is practically not required for serotonin production in the midbrain.
Gata2 expression in the midbrain is regulated by a gene proximal upstream region
To begin to understand the molecular mechanisms by which the unique expression pattern of Gata2 in the midbrain is achieved, we localized the activity of the Gata2 gene regulatory domains. As shown in Fig. 2A, the Gata2 gene harbors two alternative promoters/first exons (IS and IG; Minegishi et al. 1998). Therefore, we first examined promoter usage in the midbrain expression of Gata2 using two independent GFP knock-in mouse lines, that is, EIS-KI and EII-KI (Fig. 2A). The EIS-KI knock-in allele was designed to express GFP exclusively from the IS exon (Fig. 2C). However, the EII-KI allele was designed to recapitulate the entirety of Gata2 gene expression by inserting the GFP reporter into the translation initiation methionine codon located in exon II. In the latter case GFP expression was also detected in E11.5 midbrain (Fig. 2B). The midbrain GFP expression in the EIS-KI and EII-KI knock-in mice appeared to be virtually identical at all stages we examined (data not shown). RT-PCR analyses confirmed that both the IS and IG promoters are used for Gata2 expression in the midbrain (Supplementary Fig. S2).
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We next isolated GFP-positive cells from E10.5 midbrains of 3.1ISGFP transgenic mouse embryos by FACS, and endogenous Gata2 gene expression was quantified by RT-PCR. FACS analysis showed that 2.1% and 0.9% of total cells in the midbrain and spinal cord, respectively, were positive for GFP (Fig. 2G). This ratio is comparable to the results of immunohistochemical analyses of midbrain sections stained with an anti-GFP antibody (data not shown). The expression of Gata2 mRNA in the GFP-positive cell fraction was much higher than in the GFP-negative or total cell fractions (Fig. 2G), demonstrating that the expression of 3.1ISGFP transgene is highly correlated with endogenous Gata2 expression both in the midbrain and spinal cord.
Identification of two regulatory domains for Gata2 expression in the midbrain
To analyze further the cis-acting elements regulating midbrain expression of Gata2, the conservation of sequences corresponding to the 3.1IS region among multiple vertebrate genomes was examined using a Blast-like alignment tool (BLAT). The analysis identified two highly conserved sequence domains in the region, and we refer hereafter to the distal and proximal domains as the 5H and 2H domains, respectively (Fig. 3A). We previously investigated significance of the distal 5H domain in fetal hematopoietic cells, and identified a 26-bp regulatory element in that domain. We designated the element as the early hematopoietic cell regulatory domain of the Gata2 gene (G2-EHRD), which contains functional binding motifs for GATA factors (Kobayashi-Osaki et al. 2005).
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We also examined trans-activation potential of the proximal 2H domain by generating 2H-ISGFP transgenic mice. We found that of the 22 transgene-positive embryos, 8 displayed GFP expression in the midbrain and spinal cord at E11.5 (Fig. 3J–L). To our surprise, however, the GFP reporter transgene expression in the IC region of the midbrain was absent in all eight embryos that expressed GFP in the SC sub-region. The IC-specific absence of reporter transgene expression was also observed in the adult midbrain of 2H-ISGFP transgenic mice (Fig. 3M). Taken together, these results identified two discrete midbrain regulatory domains of the Gata2 gene. The distal 5H domain appears to direct the expression of Gata2 widely in both the IC and SC, whereas the proximal 2H domain restricts Gata2 transcription to the SC sub-region.
When GFP fluorescent intensities in the midbrain of 3.1ISGFP and 5H-ISGFP transgenic mice were quantified, there was practically no difference in the ratio of SC to IC fluorescence (Supplementary Fig. S3). The expression level of the 5H-ISGFP transgene was not reduced in the SC sub-region, even though the transgene did not contain the 2H domain (Supplementary Fig. S3). These results suggest that both 5H and 2H domains harbor activity that regulates Gata2 activity in the SC sub-region, but their functions are neither additive nor synergistic. In the SC, the 5H and 2H domains appear to redundantly activate Gata2 expression, whereas in the IC region, Gata2 expression depends exclusively on 5H activity. Thus, Gata2 gene expression in the SC and IC sub-regions of the midbrain is regulated by a combination of two distinct domains.
GATA factors regulate Gata2 gene midbrain expression
Alignment of the distal 5H and proximal 2H sequences revealed that several GATA factor binding sites are highly conserved among multiple mammalian genomes (Fig. 4A and Supplementary Fig. S4), suggesting that both the 5H and 2H domains are bound by GATA factors in the midbrain and that they activate Gata2 gene expression. Based on the conservation of those GATA sites we further surmise that Gata2 gene expression in the midbrain may be controlled in an autoregulatory manner through these two distinct domains. In this regard, our previous transgenic reporter analyses proved that GATA motifs in the distal 5H domain are necessary for the Gata2 gene expression in hematopoietic cells, and biochemical analyses provided additional evidence for binding of the 5H GATA boxes by GATA factors (Kobayashi-Osaki et al. 2005).
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In the reporter co-transfection analysis, expression of the 2H-Luc reporter was induced approximately sixfold by simultaneous expression of GATA2 (Fig. 4B). This level of activation was significantly higher than that achieved by GATA3 co-transfection. No synergistic or additive effects were detected in cells co-transfected both GATA2 and GATA3 (Fig. 4B), suggesting that GATA2 alone is responsible for activation of the Gata2 gene in neural cells, and that GATA3 cannot activate the 2H midbrain enhancer as efficiently as does GATA2. Several 2H-Luc mutants harboring mutations in the GATApal motif were also examined in these luciferase assays, but no significant change was observed in these mutants when compared to the wild-type 2H reporter (data not shown). We speculate that the other cis-acting GATA motifs (9 GATA consensus sites within the 359 bp 2H enhancer, open boxes in Fig. 4A) in the 2H sub-region may compensate for the loss of GATApal activity in these transfection assays.
GATA2 is indispensable for Gata2 transcriptional activation in the midbrain
In order to determine the role, if any, GATA2 might play in activation of the Gata2 gene, we crossed heterozygous Gata2-null mutant mice (Gata2+/–) to mice bearing the 3.1ISGFP transgene. Through subsequent breeding, the 3.1ISGFP transgene was integrated into the Gata2–/– mouse genome, so that the GFP expression in the midbrain of the Gata2–/– mouse (Fig. 5A–C) could be compared with that of wild-type mice at E10.5 (Fig. 5D–F), just before embryonic demize of the Gata2-null embryos (Tsai et al. 1994).
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We previously observed midbrain GFP expression in 3.1ISGFP transgenic embryos only after E10.5 (Kobayashi-Osaki et al. 2005), whereas midbrain fluorescence in EII-KI and EIS-KI mice could be detected by approximately E9.5 (Suzuki et al. 2006). This discrepancy suggested that the initiation of Gata2 expression in the midbrain might require some additional regulatory information that was located outside of the 3.1IS region. An intriguing question here was whether or not GATA2 is also required for the initiation of Gata2 gene expression in the midbrain. To address this question, we examined the expression of GFP in the midbrain of Gata2-null mutant mice (EII-KO) in which GFP gene was knocked into the Gata2 gene (Gata2GFP/–). The EII-KO mice were generated through crossing the EII-KI (Gata2GFP/+) mice with Gata2+/– mice. The EII-KO embryos displayed an identical embryonic lethal phenotype to Gata2–/– embryos, allowing us to conclude that the EII-KO allele is a genuine null (Tsai et al. 1994; Suzuki et al. 2006). Importantly, we detected GFP expression in the mesencephalon of EII-KO embryos at E10.0 (Fig. 5I,J). The GFP expression pattern in EII-KO embryo (Fig. 5I,J) was similar to that in EII-KI embryos at E10.0 (Fig. 5G,H), and no histological abnormality was noticed in the tissue sections of EII-KO midbrain (data not shown). These results thus demonstrate that, although GATA2 is necessary for the stable autoregulation of the Gata2 gene in the midbrain after E10.5, GATA2 itself is dispensable for the initial activation of the gene in the E9.5 midbrain.
We exploited similar approach to clarify possible compensatory roles for GATA3 in Gata2 gene expression. For this purpose, we used a Gata3 knock-in line that also generates a null allele (G3-KI, Gata3LacZ/+; Hendriks et al. 1999). The study was conducted by intercrossing the G3-KI (Gata3LacZ/+) mice with the 3.1ISGFP transgene. We found that 3.1ISGFP transgene was expressed normally in the midbrain of E11.5 embryos even in the homozygous Gata3lacZ/lacZ background (Fig. 5M,N), and the expression profile was indistinguishable from G3-KI:3.1ISGFP embryos (Fig. 5K,L). These results, taken together, indicate that GATA3 plays no role in midbrain Gata2 gene regulation.
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Discussion |
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We found in this study that GATA2 is highly expressed in the midbrain, but not in the cerebrum and cerebellum of adult mouse brain. In the midbrain, cells expressing Gata2 were distributed in the SC, IC and DRN. Although midbrain function is related to the visual and auditory sensations, details of the function are still elusive (Wallace et al. 1993; Krishna & Semple 2000). The GFP transgenic mice used in this study must be a useful means to monitor the midbrain development and also to isolate Gata2 expressing cells from the midbrain. Especially, the 2H-ISGFP transgene is available as a specific marker for the SC. GATA2 has been suggested to regulate proliferation and differentiation of immature neural progenitor cells. For instance, GATA2 plays a role in maintaining neuronal progenitor cells at quiescent status (El Wakil et al. 2006) and GATA2 is essential for development of V2 interneuron in the spinal cord (Zhou et al. 2000). On the contrary, this study revealed that virtually no morphological abnormality was observed in the midbrain of Gata2-null mice during the time the mice alive. Similarly, neural cell-specific deletion of the Gata2 gene in mice did not affect development of the midbrain (T. Hoshino and M. Yamamoto, unpublished data). These results indicate that GATA2 is dispensable for the midbrain formation even though it is specifically and highly expressed in there.
A small portion of Gata2 expressing cells in DRN are found to synthesize serotonin, and this observation is consistent with the previous finding that expression of serotonin biosynthetic enzymes in the hindbrain cells requires GATA2 (Craven et al. 2004; Alenina et al. 2006). However, most of the Gata2 expressing cells in the midbrain were negative for serotonin production, suggesting that GATA2 in the midbrain is irrelevant to the serotonin biosynthesis. As GATA3 is also expressed in the midbrain (George et al. 1994; Nardelli et al. 1999), one plausible hypothesis is that GATA3 may have an activity compensating GATA2 in the midbrain. However, cells expressing Gata2 is clearly different from those expressing Gata3 in the midbrain. Thus, our current observation does not support the hypothesis, and further studies are necessary to understand the physiological contribution of GATA2 to the midbrain formation and function. After acceptance of the present paper, a study describing Gata2-expressing midbrain cells was published (Kala et al. 2009). This study demonstrates that GATA2 is required for specification of GABAergic neurons but dispensable for proliferation of the midbrain cells. This finding is in agreement with our study showing that Gata2 gene expression is not detectable in serotonergic neurons andis not essential for morphogenesis of the midbrain.
We previously identified two first exons, that is, IS and IG exons, of the Gata2 gene (Minegishi et al. 1998). In this study we found that both exons are used in the midbrain and 3.1 kbp genomic region upstream of the IS exon (3.1IS region) can direct the midbrain expression of Gata2 after E10.5. This is consistent with our previous observation that the proximal promoter region of Gata2 is required for the midbrain expression of the gene (Zhou et al. 2000). The 3.1IS region harbors two cis-acting domains, distal 5H and proximal 2H, which are highly conserved among the mammalian genomes. Intriguingly, these two regulatory domains play different roles in the SC and IC sub-regions. In SC either 5H or 2H is sufficient to activate the Gata2 expression, whereas in IC 5H is sufficient for the gene expression but 2H is not. Thus, two cis-acting domains in close proximity individually direct the specific gene expression in two sub-regions of one organ. Consistent with this observation, we previously identified two tandem enhancers for the Gata2 expression in the urogenital system, which direct the gene expression specifically in two separated urogenital regions (Khandekar et al. 2004). Similarly, the SCL gene was reported to contain two GATA boxes that show differential contributions to the two midbrain sub-regions (Sinclair et al. 1999).
There are several GATA boxes in the 5H domain. The 5H domain also serves as an enhancer for the Gata2 gene expression in the hematopoietic cells, and some of the GATA boxes are important for the Gata2 gene expression in hematopoietic cells (Kobayashi-Osaki et al. 2005). We speculate that through these GATA motifs this domain is used commonly in the midbrain and hematopoietic cells. Similarly, the 2H domain also contains several GATA boxes including a GATApal motif, but the biological activity of the GATA boxes in the 2H domain remains to be clarified.
In this study the loss of function analysis clearly demonstrates that GATA2 is required for the Gata2 gene expression in the midbrain. GATA2 binds and activates the 5H and 2H domains in transfection analysis. Thus, GATA2 regulates its own gene in the midbrain, and the 5H and 2H domains play central roles in this autoregulatory loop. Supporting this notion, it has been reported that exogenous human GATA2 induces expression of the endogenous Gata2 gene in chick embryo spinal cord (El Wakil et al. 2006). As it is critically important to regulate strictly the expression of master transcription factors, these transcription factors often acquire regulation of their own gene in a manner adopting the positive and/or negative autoregulatory systems. For instance, positive autoregulation for the MyoD, Gata1 and PU.1 genes, and negative autoregulation for the Runx2 gene have been reported (Thayer et al. 1989; Drissi et al. 2000; Nishikawa et al. 2003; Okuno et al. 2005). This study for the first time reveals a positive autoregulation of the Gata2 gene in vivo using mouse genetics approach, implying that abundant GATA2 is required for midbrain development/function and is produced through the positive feedback system.
The expression of 3.1ISGFP transgene in the mesencephalon was observed after E10.5 (Kobayashi-Osaki et al. 2005), suggesting that the autoregulatory system of Gata2 via the 5H and 2H domains starts approximately E10.5. By contrast, the GFP expression in the midbrain of EII-KI and EIS-KI knocked-in mice begins at E9.5, suggesting that the 3.1IS region may not be sufficient for initiating of the midbrain expression of the endogenous Gata2 gene. Furthermore, GFP expression from the Gata2 locus of EII-KO mouse lacking the GATA2 expression was detected in the mesencephalon at E10.0. Thus, the regulatory mechanism of the Gata2 gene may be different in the initiation stage and maintenance stage; the autoregulatory system is working only for the maintenance stage. Apparently, the positive autoregulation system is useful to maintain gene expression at high level, so that use of the system seems to be reasonable after establishment of the initial expression. Indeed, it was recently reported that the expression of Ad4 BP/SF-1 is regulated by positive autoregulation, and before the establishment of autoregulation homeobox transcription factors act to initiate the Ad4 BP/SF-1 gene expression during adrenal development (Zubair et al. 2006).
A schematic model summarizing regulatory mechanism of the Gata2 gene in the midbrain is shown in Fig. 6. This study delineates that the Gata2 gene regulation system in the midbrain can be divided into two stages, one is the initiation stage and the other is the maintenance stage. In the initiation stage, a factor yet unknown supports initiation of the Gata2 expression in the mesencephalon of E9.5–E10.5 embryos through binding to a cis-acting element located outside of the 7.0IS region. The GATA2-dependency of the Gata2 gene expression appears approximately E10.5 after switching of the regulatory system to maintain the Gata2 gene expression at high level by the positive feedback system. In this maintenance stage, the Gata2 expression is spread in the SC and IC sub-regions. In SC, GATA2 induces the gene expression through binding to both 5H and 2H domains in the promoter proximal region. However, in IC 5H is used exclusively in the gene expression.
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The EIS-KI (Gata2IS–GFP/+ genotype) and EII-KI (Gata2GFP/+) mouse lines are as previously described (Suzuki et al. 2006). The Gata3-LacZ knock-in mouse line was as previously described (Hendriks et al. 1999) and heterozygous and homozygous mutants were referred to as G3-KI (Gata3LacZ/+) and G3-KO (Gata3LacZ/LacZ), respectively. Gata2-null mice were kindly provided by Dr Stu Orkin (Tsai et al. 1994). The EII-KI and Gata2-null mice were crossed to generate EII-KO (Gata2GFP/–) mice. 7.0ISGFP, 3.1ISGFP, and 5H-ISGFP transgenic lines were as previously reported (Minegishi et al. 1999; Kobayashi-Osaki et al. 2005). Genotyping was performed by PCR using genomic DNA extracted from the tail of adult mice or a part of embryos as templates (Kobayashi-Osaki et al. 2005).
The 0.6ISGFP transgene was prepared from p7.0ISGFP (Minegishi et al. 1999) by digesting with EcoRI (656 bp from the transcription initiation site of IS exon) and HindIII. To construct 2H-ISGFP transgene, the 2H domain (–1977 to –1619) was amplified by PCR, and resulting product was ligated to 0.6ISGFP transgene construct. Transgene constructs were isolated from the vector sequences, and purified with QIAquick Gel Extraction Kit (QIAGEN, Venlo, Netherlands). DNA (5 ng/mL) was injected into fertilized mouse eggs using standard protocols as described (Kobayashi-Osaki et al. 2005), and embryos and pups harboring the transgene were identified by PCR. All mice were strictly kept in specific pathogen-free conditions and were treated according to the regulations of ‘The Standards for Human Care and Use of Laboratory Animals of the University of Tsukuba.’
Histological analyses
Tissues and embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30–60 min at 4 °C. Fixed samples were embedded in OCT compound (Sakura Finetec, Tokyo) and rapidly frozen in liquid nitrogen, before making sections of 7 µM (for X-gal and immunohistochemical staining) or 50 µM (for immunofluorescence) in thickness.
For X-gal staining, fixed embryos or frozen sections were incubated with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (Wako, Osaka, Japan) at 37 °C in the overnight. For immunohistochemistry, frozen sections were treated with 1% hydrogen peroxide to block endogenous peroxidase activity. Before antibody staining, the samples were blocked with 1% goat serum and 0.2% bovine serum albumin in 2% skim milk and 0.1% Triton X-100 in phosphate-buffered saline (PBS-MT). The sections were incubated with rabbit anti-GFP antibody (diluted 1 : 1000, MBL, Nagoya). Specific antibody binding was visualized as brown staining by reaction with diaminobenzidine (250 mg/mL) with 0.01% H2O2 after incubation with horseradish peroxidase-conjugated anti-rabbit IgG (BioSource, Camarillo, CA). Hematoxylin was used for counterstaining.
For serotonin immunofluorescence staining, sections were incubated with rabbit anti-serotonin antibody (diluted 1 : 50, Zymed, San Francisco, CA) at 4 °C overnight followed by incubation with biotinylated goat anti-rabbit IgG antibody (diluted 1 : 1000, Vector Laboratories, Burlingame, CA) for 1 h and CY5-streptavidin (diluted 1 : 500, Zymed) for 1 h. After washing, sections were mounted with fluorescent mounting medium (Dako, Glostrup, Denmark) and examined by confocal laser scanning microscopy (Zeiss, Oberkochen, Germany).
Reverse transcription-polymerase chain reaction (RT-PCR)
RNA was isolated from the cerebrum, midbrain, cerebellum, thymus and lung of adult BDF1 mice using Isogen (Nippon Gene, Toyama, Japan) and reverse transcribed by SuperScript II RT (Invitrogen, Carlsbad, CA) and random primers. For semi-quantitative analyses, each cDNA was diluted so that the hypoxanthine phosphoribosyltransferase (HPRT) amplicon was equivalent amongst the test samples. PCR reaction (42 cycles) was performed as follows: 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, using primer pairs G2s (5'-TGCAACACACCACCCGATACC-3') and G2as (5'-CAATTTGCACAACAGGTGCCC-3') for Gata2, G3RT5' (5'-TCTCACTCTCGAGGCAGCATGT-3') and G3RT3' (5'-GTACCATCTCGCCGCCACAG-3') for Gata3 and HPRTs (5'-GTGGTGAAAAGGACCTCT-3') and HPRTas (5'-ACAGGACTAGAACACCTGC-3') for HPRT.
Quantitative PCR analysis of sorted cells
The midbrain and spinal cord samples from 3.1ISGFP transgenic E10.5 embryos (line 95, Kobayashi-Osaki et al. 2005) were dissected, and the tissues were treated with 0.05% trypsin-EDTA at 37 °C for 10 min before dissociation by pipetting. GFP+ and GFP– cells in the single cell suspensions were sorted using FACS Vantage (Becton Dickinson, Franklin Lakes, NJ) as described (Kobayashi-Osaki et al. 2005). RNA was extracted by RNeasy Mini Kit (QIAGEN) from the sorted cells, and reverse transcribed by SuperScript II RT with random primers. cDNA samples were rendered quantitative RT-PCR analysis of Gata2 mRNA using ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The sequence of primers and probe were described (Suzuki et al. 2003), and the data were normalized to expression level of rRNA (Applied Biosystems).
Comparison of genomic sequences
Conservation of genomic sequences was examined with Mouse BLAT Search in UCSC Genome Browser website <http://genome.ucsc.edu> among vertebrates, that is, mouse, rat, rabbit, human, chimp, rhesus, cow, dog, armadillo, elephant, tenrec, opossum, chicken, xenopus, tetraodon, fugu and zebrafish.
Luciferase reporter analysis
The 2H domain fragment was integrated into pRBGP3 plasmid containing the firefly Luciferase gene driven by a basic promoter from the rabbit β-globin gene (Igarashi et al. 1994) to generate 2H-Luc plasmid. Expression vectors for human GATA2 and human GATA3 under control of the elongation factor-1
promoter, pEF-GATA2 and pEF-GATA3, were as previously described (Yamaguchi et al. 1998). C1300 mouse neuroblastoma cells (Dunham & Stewart 1953) were maintained in Dulbecco's modified Eagle medium (Sigma-Aldrich, St Louis, MO) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT), 50-U/mL penicillin and 50-mg/mL streptomycin sulfate. Cells were cultured in 12-well plates, and transfected 0.9 µg of 2H-Luc or pRBGP3 with or without co-transfection of GATA factor expression vectors (50 ng) using FuGene 6 (Roche, Basel, Switzerland). Twenty-four hours after transfection, luciferase activities were determined with Dual-Luciferase reporter assay system (Promega, Madison, WI) following the instructions of the manufacturer, and 0.1 µg of pRL-TK containing the Renilla Luciferase gene (Promega) was co-transfected in each well to standardize transfection efficiency. Expression levels of GATA2 and GATA3 proteins in the cultured cells were detected by Western blotting analyses using rat anti-chicken GATA2 monoclonal antibody (RC1.1.1) (Zhou et al. 1998) and mouse anti-human GATA3 monoclonal antibody (HG3-31, SANTA CRUZ, Santa Cruz, CA).
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Acknowledgements |
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Footnotes |
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These authors have contributed equally to this article. 
* Correspondence: masiyamamoto{at}m.tains.tohoku.ac.jp |
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Received: 1 November 2008
Accepted: 12 February 2009
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