SEARCH

SEARCH BY CITATION

Keywords:

  • retinoid;
  • microarray;
  • RAR;
  • neurula;
  • anteroposterior patterning;
  • organogenesis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Retinoid signaling is important for patterning the vertebrate hindbrain and midaxial regions. We recently showed that signaling through retinoic acid receptors (RARs) is essential for anteroposterior patterning along the entire body axis. To further investigate the mechanisms through which RARs act, we used microarray analysis to investigate the effects of modulating RAR activity on target gene expression. We identified 334 up-regulated genes (92% of which were validated), including known RA-responsive genes, known genes that have never been proposed as RA targets and many hypothetical and unidentified genes (n = 166). Sixty-seven validated down-regulated genes were identified, including known RA-responsive genes and anterior marker genes. The expression patterns of selected up-regulated genes (n = 45) were examined at neurula stages using whole-mount in situ hybridization. We found that most of these genes were expressed in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. Some were expressed in tissues such as notochord, somites, pronephros, and blood islands, where retinoic acid (RA) plays established roles in organogenesis. Members of this set of newly identified RAR target genes are likely to play important roles in neural patterning and organogenesis under the control of RAR signaling pathways, and their further characterization will expand our understanding of RA signaling during development. Developmental Dynamics 232:414–431, 2005. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Vitamin A deficiency (VAD) was one of the earliest diet-related deficiencies described. VAD during embryogenesis leads to a well-characterized spectrum of developmental defects, whereas a completely lack of vitamin A during development is lethal. Research in several laboratories has shown that vitamin A is required for hematopoiesis, immune function, vision, reproduction, and embryonic patterning, growth, and differentiation of both normal and malignant tissues (Sporn et al., 1994; Kastner et al., 1995; Chambon, 1996). Intriguingly, vitamin A excess can lead to teratogenic effects in many of the same tissues that are adversely affected by VAD, particularly the heart, central nervous system (CNS), eyes, limbs, and reproductive system. This finding has led to the concept that retinoid levels must be precisely controlled during development. A large number of studies have suggested that all-trans retinoic acid (atRA) can substitute for most of the developmental requirement for vitamin A; hence, atRA is considered the active principle of vitamin A insofar as embryonic development is concerned. Embryonic RA levels are tightly regulated by the availability of synthetic enzymes such as RALDH2, which convert retinol and retinaldehyde into RA, and catabolic enzymes such as CYP26, which mediate RA breakdown (reviewed in Maden, 1999, 2002).

RA exerts most of its effects on development by regulating the activity of two classes of ligand-activated transcription factors that are members of the nuclear hormone receptor superfamily (reviewed in Mangelsdorf and Evans, 1995; Chambon, 1996; Blumberg, 1997). The retinoic acid receptors (RARs) are encoded by a family of three genes (RARα, RARβ, and RARγ) that bind to atRA with nanomolar affinity (Giguere et al., 1987; Petkovich et al., 1987). The retinoid “X” receptors (RXRs) also compose a family of three genes (RXRα, RXRβ, and RXRγ) that bind the RA stereoisomer 9-cis RA (9cRA) at nanomolar levels (Heyman et al., 1992; Levin et al., 1992) as well as several other classes of compounds at micromolar levels (Kitareewan et al., 1996; Lemotte et al., 1996; de Urquiza et al., 2000; Goldstein et al., 2003). 9cRA can activate both RARs and RXRs, whereas atRA only activates RARs. RARs act as heterodimers with RXR to bind their specific retinoic acid response elements (RAREs) in the promoters of target genes. RXRs bind to their response elements as homodimers. In the absence of ligands, the receptor dimers bind to DNA and recruit factors such as nuclear receptor corepressor (NcoR)-1 and NcoR-2/SMRT (silencing mediator of RAR and thyroid hormone receptor) to induce chromatin condensation that down-regulates transcription (Horlein et al., 1995; Chen et al., 1996; Wei, 2003). Ligand binding leads to corepressor release and the recruitment of coactivators such as CBP/p300, p300/CBP-associated factor, and SRC-1/p160 steroid receptor coactivator family members that enhance transcription by relaxing chromatin conformation (Yao et al., 1996; Leo and Chen, 2000; Wei, 2003).

Genetic and functional analyses of transgenic mice carrying single and combined mutant receptors have revealed that loss of multiple RAR genes is required to elicit defects resembling VAD phenotypes, presumably because loss of one receptor is compensated by up-regulation of another (Lohnes et al., 1994; Kastner et al., 1995). In contrast to the situation in mammals, the removal of a single RAR, RARα, in Xenopus led to an unambiguous developmental phenotype, the loss of anterior neural structures, which very much resembled RA treatment (Koide et al., 2001). We also showed that increased repression mediated by RAR was sufficient to give the opposite phenotype, an increase in the size of anterior neural tissue and the expression of anterior marker genes (Otx2, BF1, and ANF1; Koide et al., 2001) with a concomitant loss in the expression of posterior neural markers (Shiotsugu et al., 2004). These observations suggest that RARs play a pivotal role in both anterior and posterior neural tissues (Shiotsugu et al., 2004; reviewed in Weston et al., 2003).

The complementary expression patterns of the RA biosynthetic enzyme RALDH2 and the RA degrading enzyme CYP26 suggest that RA levels will be high in the trunk region and low to absent in the anterior and extreme posterior during the critical period for anterior–posterior (A-P) axis patterning (Chen et al., 2001). Regional perturbation of RA levels by modulating the expression of these enzymes predictably alters A-P patterning. For instance, overexpression of RALDH2 led to anterior extension of both midbrain and hindbrain marker genes, En-2 and Krox20, associated with a reduction of forebrain territory (Chen et al., 2001). Knockdown of CYP26 by the morpholino antisense oligonucleotide induced a similar phenomenon and extended posterior neural genes (Meis3 and Hoxb-1b) anteriorly, whereas overexpression of CYP26 suppressed the posterior genes (Kudoh et al., 2002) and led to a posterior expansion of the Otx2 expression domain (Hollemann et al., 1998). Nearly identical effects were observed by modulating the activity of RARs (Blumberg et al., 1997; Koide et al., 2001). Microinjection of a constitutively active RAR resulted in anterior truncations and posteriorization of anterior neural tissue, whereas overexpression of a dominant-negative RAR reduced the expression of posterior markers (Xlim1, N-tubulin, and Hoxb-9) and enhanced the expression of anterior markers (Otx2, XBF1, and XANF1; Blumberg et al., 1997; Koide et al., 2001; Shiotsugu et al., 2004).

RARα and RARγ mRNAs are expressed in prospective anterior tissues during the stages when Xenopus embryos are most sensitive to exogenous retinoids (Ellinger-Ziegelbauer and Dreyer, 1991, 1993; Sharpe, 1992; Pfeffer and De Robertis, 1994; Koide et al., 2001). In addition to effects on A-P patterning observed in several laboratories, abnormal RA levels cause developmental defects in tissues, including CNS, kidney, heart, and somites. Some of the genes involved in these developmental abnormalities were identified as RA-responsive genes such as the neuronal differentiation markers (X-ngnr-1, X-MyT1, and Gli3; Franco et al., 1999), GATA-1 (Bertwistle et al., 1996), the early pronephros marker Xlim-1 (Taira et al., 1994), and Thylacine1 (Thy1) in somitogenesis (Moreno and Kintner, 2004). However, no large-scale study has been conducted yet to identify the pathway of response to RAR signaling in early developmental patterning.

In this study, we used DNA microarray analysis to systematically isolate genes whose expression was altered by modulating the transcriptional activity of RARs. Embryos were treated with the synthetic RAR-selective agonist TTNPB or the inverse agonist AGN193109 during the early blastula through neurula stage, the time when Xenopus embryos are the most susceptible to treatment with exogenous retinoids (Ruiz i Altaba and Jessell, 1991; Sive and Cheng, 1991). Comparison of agonist to antagonist-treated embryos minimized the influence of endogenous retinoids on the experiment, improving the sensitivity and dynamic range of the microarray analysis. The use of Bayesian statistics allowed us to maximize the number of true positives without increasing number of false positives. The microarray results were intensively validated by quantitative real-time reverse transcriptase-polymerase chain reaction (QRT-PCR). We identified a large number of putative RAR target genes, including known RA-responsive genes, known genes that have never been proposed as RA targets, and many unknown genes. The expression patterns of the putative target genes overlapped with the known expression patterns for RARs or RALDH2. Therefore, we conclude that the genes isolated in this study are likely to play roles in neural patterning and organogenesis under the control of RAR signaling pathways.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Generation of Embryos Enhanced or Depleted in RAR Target Genes

To identify genes specifically up-regulated or down-regulated by modulating retinoid signaling, we treated embryos with compounds that selectively activate or antagonize RARs without affecting other nuclear receptors. TTNPB is known to specifically activate RARs, whereas AGN193109 blocks activation of RARs in Xenopus embryos (Koide et al., 2001; Shiotsugu et al., 2004). Neither compound has any effect on RXRs, enabling us to identify genes that are RAR-responsive. Embryo treatment began at the blastula (stage 8) stage with either the strong RAR-selective pan-agonist TTNPB (10−6 M), solvent controls, or the RAR-selective pan-inverse agonist AGN193109 (10−6 M). Embryos were harvested at the late neurula (stage 18) to identify genes responsive to retinoid treatment. Ten embryos from each treatment group were allowed to develop until stage 38 to evaluate the severity and penetrance of the phenotype observed. Embryos from treatment groups showing strong phenotypes at stage 38 were used to prepare total and polyA+ RNA for further analysis. Appropriate regulation of genes known to be up-regulated (Hoxd-1) and down-regulated (Otx-2) by RA was also verified in the RNAs from treated embryos by Northern blot analysis (data not shown).

Microarray Identification of RAR Target Genes

Microarray analysis is a very powerful approach to identify target genes (Schena et al., 1995) and already has been used in the Xenopus system (Altmann et al., 2001). We decided to use microarray analysis to identify genes up- or down-regulated in response to modulating RAR signaling. The chip we used for microarray analysis contained 21,120 normalized, sequenced neurula (stage 13–15) cDNAs (Shin et al., 2005). These 21,120 cDNAs represent approximately 8,700 distinct transcripts. Fluorescently labeled probes were generated from poly A+ RNA prepared from TTNPB- or AGN193109-treated embryos, hybridized to the microarrays, washed, and scanned.

Despite its power as a system, some technical difficulties must be addressed for the results of microarray analysis to be reliable. The first problem is that, because retinoids are present in normal embryos, there will be some background of induced mRNAs in the control embryos, reducing the sensitivity and dynamic range of the assay. To overcome this problem, we compared agonist with antagonist-treated embryos and identified how the genes identified varied compared with control embryos during the validation step. This approach improves the dynamic range of the assay compared with agonist/control and antagonist/control probe pairs. Previous experiments suggested that gastrula stage (stage 12) embryos did not show much difference in the expression of genes already known to be direct RAR targets (e.g., Hoxb-1, Hoxd-1) in response to TTNPB treatment compared with neurula (stage 18) embryos where these differences are more pronounced. Therefore, we used RNA from stage 18 embryos for microarray analysis.

Another important issue is the difference in sensitivity, labeling efficiency, and signal/noise ratio between the red dye (cy5) and the green dye (cy3) commonly used for microarray analysis. We overcame this problem by changing from direct incorporation of fluorescent nucleotides to incorporation of aminoallyl dUTP during reverse transcription followed by chemical coupling of the dye molecules following the advice of the Brivanlou laboratory (Altmann et al., 2001). The single labeling followed by chemical dye coupling ensures that the labeling efficiency in each reaction is virtually identical. Further improvement was obtained by substituting Alexa Fluor 546 for Cy3 and Alexa Fluor 647 for Cy5. This change produced very intensely labeled probes that exhibited substantial increases in the red signal and dropped background to levels comparable to the green channel making normalization of channel intensity unnecessary.

Our initial experiments used embryos from several frogs, and the resulting mRNA was pooled. The probes were prepared, labeled, hybridized to the arrays in triplicate, washed, scanned, and analyzed. The fold induction was calculated as an average ratio of median feature pixel intensity at wavelength 635 nm to median feature pixel intensity at wavelength 532 nm. Northern blotting was used to establish the validity of the microarray analysis. To our surprise, 40% of the top 35 up-regulated genes (12 of 29) by microarray were not changed when checked by Northern blot (data not shown). Such a false-positive rate is incompatible with identifying the network of target genes regulated by RARs; therefore, we modified the approach to reduce false positives.

It has been suggested that biological variation is the largest source of error in microarray experiments (Baldi and Hatfield, 2002). We reasoned that embryos from an individual mating would exhibit stochastic fluctuations in gene expression that would be different from those in matings between different males and females. Therefore, embryos were obtained from matings between three different pairs of males and females and subsequently kept strictly separate and treated independently. Probes were prepared from the RNA populations isolated from treated groups and used as matched pairs derived from the same frog in competitive hybridization to the microarray. Each experiment was replicated yielding a total of six data sets. Bad hybridization spots were filtered from each data set before statistical analysis. Thus, the total number of replicates varies by clones depending on hybridization quality, even though all experiments were performed six times. By using this approach and calculating fold-induction values for each agonist/antagonist pair, none of the true positives from the first experiment was lost, whereas all of the false positives disappeared.

To further improve the statistical validity of our results, we used Cyber-T software, which was developed by Pierre Baldi, Tony Long, and colleagues at UCI (http://visitor.ics.uci.edu/genex/cybert/ Baldi and Long, 2001; Long et al., 2001). Cyber-T analysis allows users to chose either the observed variance among replicates within treatments (simple t-test) or a Bayesian estimate of the variance among replicates within treatments based on a prior distribution obtained from a local estimate of the standard deviation (Bayesian statistics). We first tested which method generated the largest number of expressed sequence tags (ESTs) showing statistically significant changes in expression in response to ligand treatment. P values from Bayesian statistics were plotted against log transformed fold changes (magnitude of gene expression, Fig. 1). A sliding window size of 101 and a Bayesian confidence value of 10 were used. The number of statistically significant clones (P < 0.01) identified was 3,759 for the simple t-test compared with 4,811 by Bayesian analysis. The smallest fold change observed in the Bayesian analysis was 1.18-fold, whereas it was 1.08-fold for the simple t-test. Therefore, Bayesian analysis is not expected to increase the number of false positives and this expectation proved to be the case when we tested the response of these genes by Northern and QRT-PCR analysis (see below).

thumbnail image

Figure 1. Relationship between fold change and magnitude of the gene expression change as a function of the Bayesian statistical significance for all 18,437 clones. Significance is plotted as the negative log10 of the P value, and the magnitude represents the log base two-fold change in gene expression. The ordinate axes show arbitrary cutoff of 1.75-fold for down-regulated clones and 1.5-fold for up-regulated clones, and the secondary abscissa axis shows P value of 0.01. The points in section a represent clones that are changed less than arbitrary cutoff but are statistically significant. The points in section b represent clones that are changed less than the arbitrary cutoff and are not statistically significant. The points in section c represent clones both statistically significant and altered by more than the arbitrary cutoff. The points in section d represent clones that are changed more than arbitrary cutoff but are not statistically significant.

Download figure to PowerPoint

We chose 1.5-fold up-regulation and P < 0.01 as initial criteria for determining significance. Samples that had less than three repetitions were automatically eliminated during data analysis by Cyber-T. The resulting data set consisted of 18,437 ESTs, which have three to six repetitions. The number of up-regulated clones predicted to be significant by the simple t-test was 505, whereas Bayesian statistics yielded 571. Thus, 66 ESTs shown to exhibit significant changes using Bayesian analysis were not statistically significant using the simple t-test. Among these 66 ESTs are two known to be up-regulated by RA: XCYP26 (Hollemann et al., 1998) and XMeis1 (Mercader et al., 2000). We sampled these 66 ESTs and found that 18 of 19 were validated by QRT-PCR and 3 of 3 by Northern blotting. Overall, these results show that Bayesian statistics detected more true positives than simple t-statistics without increasing the number of false positives. Therefore, Bayesian statistics were used for all data analysis in this manuscript.

To more confidently estimate the minimum fold change required for a gene to be considered significant, we sampled a subset of the putative up-regulated and down-regulated genes and verified that they were regulated in embryos treated with ligands. Northern blot analysis was performed with selected genes, including known RA-responsive genes, anterior and posterior marker genes, and uncharacterized genes (Fig. 2). The expression levels of RA up-regulated genes such as midkine (MK) that was enhanced by RA treatment in mammals (Muramatsu and Muramatsu, 1991), RA degradation enzyme CYP26 (Hollemann et al., 1998), short-chain dehydrogenase/reductase (SDR; Cerignoli et al., 2002), posterior marker genes, Xcad3 (Shiotsugu et al., 2004), XMeis3 (Kudoh et al., 2002), and Forkhead transcription factor HNF-3α (Jacob et al., 1994) were enhanced with TTNPB treatment and reduced with AGN193109 treatment. Meanwhile, RA down-regulated anterior marker genes such as Otx-2 (Pannese et al., 1995) and XA-1 (Hemmati-Brivanlou et al., 1990) behaved as expected in response to ligand treatments. All tested clones, including unknown and hypothetical genes, altered their expression in response to ligand treatment in the same direction in Northern blotting or QRT-PCR as in the microarray results. As Northern blot analysis is not suitable for testing the expression of large numbers of genes, we adopted QRT-PCR as our main gene expression analysis tool and compared the expression of selected genes in TTNPB, AGN193109 and solvent treated embryos. We repeated validation of Xcad-3, XMeis3, Fetuin B, MK, HNF-3α, and two ESTs (XL044f02 and XL041b23) in QRT-PCR and confirmed that QRT-PCR generated similar results to those of Northern blot analysis. A total of 113 up-regulated and 115 down-regulated genes were tested by QRT-PCR and/or Northern analysis. Table 1 shows overall QRT-PCR results.

thumbnail image

Figure 2. Northern blot analysis. Equal amounts of total RNA isolated from controls or embryos treated with TTNPB or AGN 193109 were electrophoresed, blotted to nylon membranes, and hybridized with probes derived from each clone. The name of each clone used for probe-templates and its closest match found in public databases are given on the left of each panel. Fold and (PCR) indicate change in microarray analysis and a validation result of quantitative real-time reverse transcriptase-polymerase chain reaction (QRT-PCR). C, T, and 109 indicate control, TTNPB, and AGN193109 treatment, respectively. The asterisks indicate known RA target and/or posterior marker genes, and ISH indicates clones used as probes for whole-mount in situ hybridization. MK, Midkine; VLCS, very-long-chain acyl-CoA synthetase; SDR1, short-chain dehydrogenase/reductase; LCS, long-chain acyl-CoA synthetase.

Download figure to PowerPoint

Table 1. Validation of Putative RAR Target Genesa
 Number of genesTested (QRT-PCR)Validation
  • a

    RAR, retinoic acid receptor; QRT-PCR, quantitative real-time reverse transcriptase-polymerase chain reaction.

Up-regulated genes fold   
 >1.910239100% (39/39)
 1.6∼1.91303795% (35/37)
 1.5∼1.61302483% (20/24)
 Total36210094% (94/100)
Down-regulated genes fold   
 >2.0956766% (44/67)
 1.75∼2.01032361% (14/23)
 > 1.751989064% (58/90)
 1.5∼1.754042536% (9/25)

The genes were divided into three groups based on fold induction and a fraction of genes in each group was validated by QRT-PCR (Table 1) and/or Northern blot analysis (100%, 12 of 12 in group 1, 6 of 6 in group 2, and 2 of 2 in group 3). Of these genes validated by Northern blot analysis, 7 of 7 were also validated by QRT-PCR. Overall validation rate was 92%, which was calculated by the following formula (102 × 1 + 130 × 0.97 + 130 × 0.83)/362 × 100 = 92%. Therefore, we performed clustering analysis on the entire group of up-regulated genes.

The down-regulated gene set was much less robust than the up-regulated set. We set a significance threshold of 1.75-fold, P < 0.01 because the dimethyl sulfoxide–negative controls on the microarray appeared at 1.75-fold down-regulation. The down-regulated set was further subdivided into three groups, although there was no dramatic difference in the validation rate between these groups (Table 1). The validation of genes showing more than 3.0-fold change was 3 of 3 (100%) by Northern blot analysis. Considering the much lower rate of validation compared with the up-regulated genes, we limited cluster analysis to the subset of validated genes. However, it should be noted that, although genes below the selected thresholds validate at relatively low frequency, a significant fraction (36%) does validate and may be biologically important.

Cluster Analysis of Putative RAR Regulated Genes

The ESTs selected above were clustered into putative unique genes based on sequence similarity using the original EST sequences and clustering available in XDB (http://Xenopus.nibb.ac.jp) as well as the Xenopus UniGene set (http://www.ncbi.nlm.nih.gov/UniGene/UGOrg. cgi?TAXID=8355) and used for similarity searches in public database. Sixty clones present on the microarray that did not have sequence information in XDB were recovered and sequenced from the 5′-end and used for the annotation. As we expected, a subset of the ESTs were apparently derived from the same genes. The 571 up-regulated clones (≥ 1.5-fold, P < 0.01) were clustered to 362 putative unique genes. The 303 down-regulated clones (≥ 1.75-fold change, P < 0.01) were clustered to 197 putative unique genes. It should be noted that the number of significantly changed genes might be further reduced as sequence information is obtained for those ESTs present on the microarray that do not have sequence entries in the database.

Because the microarray was prepared from randomly picked ESTs from a normalized cDNA library, one expects some degree of duplication in the genes represented. Detection of genes present at multiple locations on the array provides a measure of internal consistency for the overall experiment and many genes that were highly up-regulated were detected more than once (Table 2). The RAR-regulated genes were manually clustered according to the published literature (Fig. 3A,B). Before cluster analysis of the up-regulated genes, those that did not validate by QRT-PCR (n = 6) and those lacking sequence information (n = 22) in the database were excluded from further consideration (n = 334 in total). The majority of up-regulated genes are hypothetical (similar to ESTs from other organisms) or unidentified (did not show significant similarity to database sequences from any organism). There are transcription factors, including multiple homeodomain proteins, nuclear receptors, GATA transcription factors and RA-inducible proteins, neurogenic proteins, and other tissue development factors. The identity and function of up-regulated and down-regulated genes are provided in Supplemental Tables 3 and 4 (which are available online at http://www.interscience.wiley.com/jpages/1058-8388/suppmat).

Table 2. Frequency of Occurrence of Up-regulated Clones and Their Average Expression Levels
FrequencyNumber of genesNumber of clonesAvg. expression
1x265265 (73.2%)1.76
2x51102 (14.1%)1.96
3x2369 (6.4%)2.64
4x1040 (2.8%)2.33
5x420 (1.1%)1.80
6x212 (0.6%)2.33
7x17 (0.3%)5.16
8x432 (1.1%)1.86
9x19 (0.3%)7.43
15x115 (0.3%)2.32
Total362571 (100%)2.11
thumbnail image

Figure 3. Classification of genes regulated by RARs. Up- and down-regulated genes were categorized according to the predicted or established functions of translated products acquired by a similarity search against DNA and protein databases. A: Upregulated genes (n = 334) were functionally divided into 17 categories; β-oxidation, cytoskeleton, DNA modification, extracellular matrix, gluconeogenesis/glycolysis, housekeeping, hypothetical, miscellaneous, mitochondrial, neural, protein metabolism/remodeling, RA degradation, signal transduction, transcription, transporter/exchanger, tumor suppressor and unidentified. B: Down-regulated genes (n = 67) were functionally divided into 10 categories; cytoskeleton, extracellular matrix, housekeeping, hypothetical, miscellaneous, neural, RA synthesis, signal transduction, transcription, and unidentified.

Download figure to PowerPoint

Expression Pattern of RAR Target Genes

We determined the expression patterns of 43 up-regulated genes encoding hypothetical/unidentified proteins in stage 18 and 22 embryos by whole-mount in situ hybridization. We also characterized the expression of two Xenopus genes encoding thyroid hormone receptor TR-βA and U8snoRNA. A significant portion of the clones tested (37/45) showed staining in neural tissue; nine genes exhibited expression in the brain, and 13 showed neural crest expression. Expression patterns of the genes tested are summarized in Table 5. The eye anlagen was a site of expression for 22 different genes, and somite expression was seen in 16 genes. Five genes showed strong staining in the lateral plate mesoderm, which is the major site of RALDH2 expression during neurula stages. Other notable staining patterns included the blastopore, cement gland, presumptive pituitary anlagen, pronephros, and blood islands. We were rather surprised to see that every clone tested exhibited a distinct expression pattern in either stage 18 or stage 22 embryos.

In cases where it was not possible to discern from whole-mounts which tissues exhibited staining, we examined transverse sections of the embryos (n = 23). Figure 4 shows sample staining of the somites (Fig. 4Q3), neural tube (Fig. 4N3), roof plate of the neural tube (Fig. 4M3), the neural tube and somites (Fig. 4P3), the lateral neural tube (Fig. 4O3), and the notochord (Fig. 4R3). The staining patterns in the whole and sectioned embryos were summarized and used to categorize genes broadly into four groups by expression pattern (Supplemental Table 5). The staining patterns by which we chose to divide the genes were the neural tube (nt), neural crest (nc), brain (br), eye anlagen (eye), notochord (no), somites (sm), lateral plate mesoderm (mes), and others, including pronephros (pr), blood islands (bi), cement gland (cg), and pituitary anlagen (pa). The signature staining for group 1 (n = 12) was present in the neural tube, neural crest, and optic anlagen. Examples of group 1 (XL027f19 and XL010b24) showed predominant neural crest and eye anlagen staining with or without staining in the closing neural tube at stage 18 (Fig. 4A1,A2,B1,B2). Both expression patterns were similar in stage 22 embryos with the migrating neural crest, eye anlagen, and neural tube staining (Fig. 4A3,A4,B3,B4). Although the examples did not show brain expression, two genes (XL021i22 and XL037p18) included staining in the probable brain at stage 22 (Supplemental Figure). XL010g16 expression included staining in the brain and neural tube at stage 18 and 22 (Supplemental Figure). Somite expression was detected in 8 genes in this group. Three genes had staining in pituitary anlagen (data shown in Supplemental Figure), and one was expressed in the presumptive pronephros and the ventral blood islands (Fig. 4C3,C6,C7). Transverse sections of the embryos disclosed that expression of two genes showing neural tube staining was localized to the presumptive roof plate (Fig. 4C5,M3) is shown in Figure 4. XL049b08 expression included staining in presumptive otic vesicle, pronephros, blood islands, and pituitary anlagen in addition to the neural tube and strong eye staining at stage 22 (Fig. 4C). This gene was clustered into this group based on the probable neural crest staining seen at stage 18 (Fig. 4C2), although the staining was unclear at stage 22. Xenopus TR-βA expression was indistinct at stage 18 but stained the neural tube, eye anlagen, neural crest, and somites at stage 22. The function of TR-βA during the neurula stage is currently unknown, because the ligand for this receptor, thyroid hormone, is not produced until much later in development.

thumbnail image

Figure 4. Expression patterns of selected retinoic acid receptor (RAR) target genes. Expression pattern of selected upregulated genes at stage 18 and stage 22 were determined by whole-mount in situ hybridization. Embryos were hybridized with probes made from indicated expressed sequence tag (EST) clones. The staining patterns in whole and sectioned embryos were summarized and used to categorize the genes broadly into four groups (group 1, 2, 3, and 4) by expression patterns. The signature staining of group 1 is present in the neural tube, neural crest, and optic anlagen; the staining patterns of group 2 are similar to those of group 1 but absent from the neural crest. Group 3 genes show staining in the neural tube but not in the optic anlagen. Group 4 genes have staining in the lateral plate mesoderm where RALDH2 is predominantly expressed. A, B, and C are in group 1; D, E, and F are in group 2; G, H, and I are in group 3; and J, K, and L are in group 4. A1–L1, A3–L3, M1–R1, and M2–R2: dorsal view; A2–I2 and A4–I4: anterior view; C6 and J2–L2: lateral view; C7: ventral view; C5: J4–L4 and M3–R3: sagittal section of stage 22 embryos. VLCS, very-long-chain acyl-CoA synthetase; Hypothetical, similar to ESTs from other organisms; Unidentified, no significant similarity to database sequences from any organism; Sm, somites; rp, roof plate of the neural tube; no, notochord; nt, the neural tube.

Download figure to PowerPoint

Group 2 (n = 10) staining is similar to that of group 1 but lacking in neural crest staining. For example, two clones encoding hypothetical proteins (XL016p20 and XL043c08) were expressed in overall neural tube and regionalized brains and regional eye anlagen in both stage 18 and stage 22 embryos (Figs. 4D, 5E). Because the expression patterns of XL016p20 and XL043c08 are remarkably similar to each other at stage 18 and 22, they may be under the control of the same regulatory pathway. XL005g23 was expressed in anterior neural tissue as regionalized brain staining in stage 18 embryos and showed strong anterior neural tube staining with probable brain and weak regional eye staining patterns at stage 22 (Fig. 4F). All genes in this group are hypothetical or unidentified proteins (Table 5).

thumbnail image

Figure 5. The effects of modulated retinoid signaling on expression pattern of retinoic acid receptor (RAR) target genes. The effects of AGN 193109, TTNPB, or vehicle control on expression pattern of three expressed sequence tags (ESTs), XL016p20 (S1–S6), XL049b08 (T1–T6), and XL043c08 (U1–U6), were determined in stage 18 embryos by whole-mount in situ hybridization. AGN193109 treatment reduced posterior neural expression of all three genes compared with controls (S1 vs. S2, T1 vs. T2, and U1 vs. U2) but increased medial staining of XL016p20 (S1 vs. S2), XL049b08 staining in the eye anlagen (T4 vs. T5), and XL043C08 expression in the forebrain (U1 vs. U2). In contrast, TTNPB treatment increased expression of XL016P20 throughout the embryo (S3, S6 vs. S2, S5) and reduced Xl043C08 staining in the forebrain and presumptive trigeminal ganglion (U3, U6 vs. U2, U5). S1–S3, T1–T3, and U1–U3: dorsal view; S4–S6, T4–T6, and U4–U6: anterior view; TTNPB, TTNPB-treated embryos; Control, vehicle control embryo; AGN193109, AGN193109-treated embryos.

Download figure to PowerPoint

Group 3 genes (n = 13) showed neural tube but no eye staining. The expression patterns in group 3 are more variable than those in groups 1 and 2. For example, XL035a11 had very strong staining in the cement gland, neural tube, and probable hatching gland, whereas another gene (XL033j21) that showed significant staining in the cement gland had neural tube and regional staining in the presumptive brain but no staining in the hatching gland (Figs. 4G, 5H). XL038o17, encoding Xenopus U8snoRNA (GenBank accession no. AF375054), showed unique expression in the neural tube and in a punctate pattern in the epidermis (Fig. 4I). Although this punctate staining appears to be excluded from the epidermis overlying the brain and neural tube, transverse sectioning of embryos revealed neural tube staining in stage 22 embryos (Supplemental Figure). Additional staining patterns seen in Group 3 included the neural crest (n = 1), somites (n = 3), brain (n = 2), and blood islands (n = 1). The fourth group (group 4; n = 5) showed significant staining in the lateral plate mesoderm where RALDH2 is predominantly expressed (Fig. 4J–L). XL010k11 (Fig. 4J) and XL053l12 (Supplemental Figure) showed somite staining in addition to the lateral staining. Three genes could not be classified into any of these four groups; these did not have neural tube staining but showed staining in the somites (XL040i08; Fig. 4Q), somites and presumptive blood islands (XL04e24; Supplemental Figure), and the notochord (XL038g12; Fig. 4R).

The similar staining patterns of several of these unidentified genes gave us some concern that these might be nonoverlapping segments of the same gene. To minimize this possibility, we extensively checked the available sequences of these ESTs against each other to rule out that they had been misclassified by the clustering programs. We also compared them with the NCBI and NIBB databases regularly up until submission of the manuscript to facilitate correct classification. The results suggest that all of these clones represent unique sequences; however, we cannot fully eliminate the possibility that some may ultimately represent different regions of the same transcript. The emerging X. tropicalis genome sequence will aid in resolving this issue.

Lastly, we tested whether treatment with TTNPB vs. AGN193109 elicited regional differences in staining for the putative RAR-responsive genes. Treatment with TTNPB caused posteriorization of neural tissues together with loss of anterior tissues and anterior neural markers. AGN193109 treatment caused loss of posterior neural markers and increased expression of anterior neural markers accompanied by enlarged heads (Koide et al., 2001). We chose XL049b08 from group 1, because its normal expression included pronephros and blood islands in addition to the neural tube and eye anlagen. XL016p20 and XL043c08 were chosen from group 2, because they were expressed in the neural tube and the anterior neural tissues in very similar patterns. As we expected, posterior neural expression of all three genes was reduced or eliminated by AGN193109 treatment compared with controls (cf Fig. 5S1, T1, U1, and S2, T2, U2) in stage 18 embryos. AGN193109 treatment led to increased medial staining of XL016p20 (Fig. 5S1 vs. S2) and expanded expression domains in the forebrain for XL043C08 (Fig. 5U1 vs. U2). In contrast, TTNPB treatment led to apparently increased expression of XL016P20 throughout the embryo (Fig. 5S3, S6 vs. S2, S5). TTNPB treatment led to a complete loss of Xl043C08 staining in the forebrain and presumptive trigeminal ganglion (Fig. 5U3, U6 vs. U2, U5). These results indicate that RAR activation is essential for the posterior neural expression of XL016p20 and XL043c08 but not in anterior neural tissues. XL049b08 expression was enhanced in the eye anlagen by AGN193109 treatment (Fig. 5T4 vs. T5) and was expanded to the anterior neural region by TTNPB with apparent loss of regional boundaries (Fig. 5T3, T6 vs. T2, T5). The posterior expression domain and the ventral expression domain were apparently unaffected in XL049b08 (data not shown). Thus, XL049b08 expression is probably regulated by RAR-mediated repression in the anterior neural tissues but RAR is not likely the dominant controller in the pronephros and blood islands.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Statistical Analysis of Microarray Data

To extract the maximum information possible from a microarray experiment, it is critical to have a robust data set wherein the fold change in gene expression is reliable and reproducible. A fundamental problem in microarray analysis is the limited replication of experiments, which tends to cause poor estimates of variance and a correspondingly poor performance of the t-test. By using the standard t-test approach, genes showing high fold changes between experimental conditions with high variability within treatments are not considered significant. A commonly used alternative approach to the t-test is ignoring the treatment variance and only considering genes that exhibit greater than two- or threefold change for further investigation. This approach has the implicit risk of excluding genes that show modest fold changes but whose differential expression pattern is highly reproducible. We used Bayesian statistical analysis in this work, as it has been shown previously to improve the robustness of microarray analysis (Baldi and Long, 2001; Long et al., 2001). This statistical method using weighted variance reduced variability of genes showing high fold changes between experimental conditions and incorporated more true positives without reducing the ability to distinguish chance occurrence from biologically meaningful data.

Cluster Analysis of RAR Target Genes

We undertook to identify genes that are specifically up-regulated and down-regulated by retinoid signaling to better understand the function of retinoids and retinoid receptors during early vertebrate development. We identified a large number of putative RAR target genes, including those known to be regulated by RA signaling (XCYP26, XLFB3 [Demartis et al., 1994], Meis3 [Aamar and Frank, 2004], Meis1 [Mercader et al., 2000], follistatin [Hemmati-Brivanlou et al., 1994], Hoxa-1 [Kolm and Sive, 1995], Xgbx-2 [von Bubnoff et al., 1996], Hoxd-4 [Moroni et al., 1993], MAP kinase phosphatase X17C [Mason et al., 1996], Xepsin [Yamada et al., 1999], XGATA-4, tubulin β-4, Xhh4 [Hochgreb et al., 2003], MK [Muramatsu, 1993], XSRC-3 [Kim et al., 1998], Sox-3 [Zygar et al., 1998], xGCNF [Barreto et al., 2003], and Xcad3 [Shiotsugu et al., 2004]) and those that could be reasonably predicted to be regulated by RAR signaling such as ortholog of human SDR1, which is induced by RA in human cells (Cerignoli et al., 2002), the cement gland specific markers XAG-2 and XAG precursor (Aberger et al., 1998) and a group of genes for which nothing previously was known regarding their relationship to RAR signaling. It should be noted that not all known RA-responsive genes are present in the Xenopus EST microarray we used. This finding explains why our results lack some known RA target genes, such as bFGF and HNF-4.

The genes we identified as RAR targets can be broadly divided into several groups for the purposes of discussion. The first group contains genes known to be regulated by retinoid signaling that were included on the chip. Of the ones that we can readily identify, 31 of 31 were regulated appropriately according to our microarray studies (although one did not validate by QRT-PCR). These genes include known up-regulated genes (shown above) and down-regulated genes such as XA-1, Xl-fli, Otx-2, Frzb-1, RALDH2, goosecoid, Pitx1, derriere, BMP-7, Sall1, and LIM-1b. This finding demonstrates that our strategy for identifying the gene expression pathways regulated by RAR signaling is reasonable and likely to be successful in uncovering new genes that are normally regulated by retinoid signaling during development. The second group contains genes that were identified in Xenopus or in other species but have not been considered as RA target. These include XER81 (Chen et al., 1999; Munchberg and Steinbeisser, 1999), GATA proteins (XGATA-2, XGATA-4, and XGATA-5b; Tsai et al., 1994; Jiang and Evans, 1996), LIM class transcription factors LOM2 and 4 (Mead et al., 2001), XId2 (Wilson and Mohun, 1995), NDRG1 (Kyuno et al., 2003), and XFFr1A (Dreyer and Ellinger-Ziegelbauer, 1996). The third group comprises unidentified or hypothetical genes. We will first discuss the known genes identified in this study and then the expression patterns of the hypothetical and unidentified genes predicted to be RAR responsive. We note that, although it is not possible to conclude from our data that these genes are regulated by RARs in all the tissues where they are expressed, their regulation by RAR signaling as shown by microarray and QRT-PCR suggests that retinoid signaling will play an important role in at least a subset of these tissues.

Effects on Genes Involved in A-P Patterning

Fibroblast growth factor (FGF), Wnt, and RAR signaling pathways are required to activate a series of posterior fate genes and suppress anterior fate genes in the neural tissues, thereby patterning the posterior hindbrain and anterior spinal cord (Isaacs et al., 1992; Pownall et al., 1996; Blumberg et al., 1997). Our previous study showed that temporal crosstalk between RAR and FGF signaling controls the embryonic posteriorization pathway (Shiotsugu et al., 2004). It appears that RA levels are tightly controlled by the elegant regulation of both RA synthesis and RA degradation. Chen et al. (2001) showed that XRALDH2 expression was localized to the presumptive presomitic and lateral plate mesoderm in the mid-trunk with a sharp anterior border at the level of the hindbrain/spinal cord boundary. In contrast, XCYP26 staining persisted in the anterior presumptive midbrain and forebrain neurectoderm and in the most posterior region of the embryos during gastrulation. The complementary expression patterns of XRALDH2 and XCYP26 suggests that RA levels will be high in the trunk region and low to absent in the anterior and extreme posterior of the Xenopus embryo. Other studies showed that RA treatment or microinjection of an mRNA encoding a constitutively active RAR caused posteriorization of neural tissues at low concentration and anterior truncation at high concentration, together with enhancement of hindbrain and some posterior markers (Blumberg et al., 1997). In contrast, down-regulating RAR signaling by microinjecting mRNA encoding a dominant-negative RAR (dnRAR) caused anteriorization of the hindbrain and enhanced expression of anterior marker genes (Blumberg et al., 1997; Koide et al., 2001). Therefore, we predicted that treatment with TTNPB would induce posterior fate genes, whereas treatment with AGN193109 would reduce the expression of posterior genes while enhancing the expression of anterior genes. As expected, TTNPB treatment led to up-regulation of posterior fate genes such as Hox genes, HNF-1B/LFB3, Meis3, and Xcad-3; and down-regulation of anterior marker genes, including Otx-2, Delta-2, XA-1 (Hemmati-Brivanlou et al., 1990), cement gland-specific genes, and XAG-2. This finding confirmed that our strategy worked effectively to cluster RAR target genes.

Two genes found in the up-regulated list are likely to be important in feedback regulation by RA during A-P patterning. The first is the Xenopus orphan nuclear receptor, germ cell nuclear factor (xGCNF). xGCNF is expressed at the end of gastrulation, reaches maximum levels during neurulation, and is expressed in an anterior to posterior gradient (Barreto et al., 2003). xGCNF is a transcriptional repressor that reduces the expression of the RA catabolic enzyme CYP26, and xGCNF expression is also enhanced by RA treatment (Barreto et al., 2003). Overexpression of GCNF led to effects on somitogenesis and tail differentiation (David et al., 1998), which resembled the effects of late application of exogenous RA (Ruiz i Altaba and Jessell, 1991). The positive feedback regulation between GCNF and RAR may be important for the regulation of RA levels during A-P patterning. The second gene, XER81, is a member of the Ets-transcription factor family and is known to be regulated by FGF signaling (Chen et al., 1999; Munchberg and Steinbeisser, 1999). Dorsal overexpression of ER81 caused partial loss of the eyes, whereas ventral overexpression led to the formation of ectopic tail-like structures (Chen et al., 1999). It was recently shown that regulation of the Xcad-3 promoter by FGFs and Wnts depended in large part on Ets binding sites, and ER81 was identified as a candidate factor (Haremaki et al., 2003). We recently showed that expression of Xcad-3 is dependent on RARα, acting both upstream and downstream of FGF signaling (Shiotsugu et al., 2004). Although XER81 had not been identified previously as an RAR target gene, the identification of it as such here suggests that ER81 may be a critical mediator of the interactions among RAR, FGF, and Wnt signaling in patterning the embryonic axis.

Development of the Pronephros

RA is an essential factor for nephrogenesis. For example, a 50% reduction in the circulating vitamin A concentration reduced the numbers of nephrons in mouse fetuses (Lelievre-Pegorier et al., 1998). Mutant mice lacking RARα1, RARα2, and RARβ2, exhibited reduction in numbers of nephrons and ureteric bud branches (Mendelsohn et al., 1994). Treatment with activin A and RA induced pronephric tubules and glomerulus in Xenopus animal cap ectoderm (Moriya et al., 2000). RA treatment enhanced the earliest known marker genes of pronephric development, Xlim-1, a LIM class homeobox gene expressed in Xenopus (Chan et al., 2000) and expression of a dominant negative RAR blocked Xlim-1 expression (Blumberg et al., 1997). We identified three genes that were up-regulated by TTNTB and down-regulated by AGN193109 treatment in our study that are potentially involved in pronephros development. The first gene is Xenopus Id2, a member of the Id (inhibitor of differentiation/DNA binding) class of helix-loop-helix protein (Wilson and Mohun, 1995). XId2 was predominantly expressed in the developing pronephros in addition to the neural tissues, including eye and brain (Wilson and Mohun, 1995; Liu and Harland, 2003). The expression of XId2 was disrupted by the bone morphogenetic protein (BMP) antagonist noggin but was not affected by FGF8 and BMP-4 (Liu and Harland, 2003). Nothing has been reported about the role of XId2 in pronephric development. The other genes, midkine (MK), a heparin-binding cytokine, and N-myc downstream-regulated gene 1 (NDRG1; Kyuno et al., 2003) appear to play roles in nephrogenesis. Vilar et al. (2002) showed that RA induced MK in metanephros from 14-day rat embryos and that nephrogenesis was significantly inhibited in the presence of neutralizing antibodies for MK in vitro. MK was initially described as an RA-inducible molecule expressed during mouse embryogenesis (Michikawa et al., 1993) and was considered to play important roles in fetal nervous system development and neurogenesis (Michikawa et al., 1993). Consistent with its purported role in other species, Xenopus MK (XMK) expression was observed in the neural tissues in early neurula to tail bud stage (Sekiguchi et al., 1995). MK expression in pronephros has not been reported. Kyuno et al. (2003) reported that overexpression of XNDRG1 led to both pronephric and somatic defects, whereas depletion of XNDRG1 using antisense morpholino oligonucleotides resulted in failure of pronephric development (Kyuno et al., 2003). XNDRG1 was expressed in the pronephros, eye, and branchial arches during neurula stage. Until this report, XNDRG1 has not been considered to be an RA target.

Blood and Heart Development and the Relationship of Retinoid Signaling to the Significant Factors in These Processes

We note that three GATA genes (GATA-2, GATA-4, and GATA-5b) were up-regulated by TTNPB treatment and down-regulated by AGN193109. GATA-4 and GATA-5 along with GATA-6 proteins are important for cardiac development (Jiang and Evans, 1996), whereas GATA-2 as well as GATA-1 is essential for hematopoietic development in mice (Tsai et al., 1994). Xenopus GATA-2 is abundantly expressed in embryos and involved in ventral mesoderm formation by suppressing Wnt-8 expression (Sykes et al., 1998). Xue et al. reported that FGF signaling inhibited BMP-4–induced erythropoiesis by suppressing GATA-2 expression (Xu et al., 1999). Bertwhistle et al. documented that RA did not affect expression of GATA-2 in ventral mesoderm (Bertwistle et al., 1996). In contrast, our data show that expression of GATA-2 mRNA was up-regulated by TTNPB and down-regulated by AGN109 by both microarray and QRT-PCR. The maternally inherited CCAAT box transcription factor (CBTF) that was proposed as a candidate enhancer of GATA-2 expression in Xenopus (Orford et al., 1998) was not regulated by RAR signaling pathway in our study. GATA-2 is expressed in the ventral blood island precursor cells at stage 19 and in ventral blood island and dorsolateral plate at later stages (23 to 28; Bertwistle et al., 1996). CBTF subunit 122 (CBTF122) was expressed in the blood islands and also the neural tube, neural crest, somites, anterior neural tissue, including eyes and brain but not cement gland between stages 19 to 28 (Orford et al., 1998). We note that expression of an up-regulated gene, XL049b08, was similar to CBTF122 in that staining was seen in the ventral blood island and neural tissue. However, XL049b08 staining in ventral blood island was not affected by RAR modulation, whereas neural staining was significantly affected by RAR modulation (Fig. 4T1–T6). Thus, XL049b08 is probably not involved in RAR-mediated blood development but may have a role in neural development under the control of RAR.

We found that two LIM class transcription factors (LMO2 and LMX1b) were down-regulated and that another (LMO4) was up-regulated by modulating RAR signaling. LMX1b was shown previously to be upstream of Wnt1 during chick CNS development (Adams et al., 2000) and to play an important role in FGF8 and Wnt1 feedback regulation during mouse isthmic maturation (Matsunaga et al., 2002). A recent study showed that activin A-mediated up-regulation of Xenopus LMX1b was suppressed by RA in a dose-dependent manner (Haldin et al., 2003) and RA is known to down-regulate the expression of GATA-1 in early Xenopus embryos (Bertwistle et al., 1996), which is consistent with our results. LMX1b expression was detected in the neural tube in embryos and in eye, brain, muscle, and mesonephros in tadpoles. Down regulation of LMO2 is also logically reasonable, because it is an essential cofactor of GATA-1 in erythropoiesis (Osada et al., 1997; Wadman et al., 1997; Mead et al., 2001). XLMO2 expression included extensive staining in the nascent ventral blood islands, the tail bud region, and the dorsal lateral plate mesoderm at stage 21 (Mead et al., 2001). Meanwhile, XLMO4 up-regulation by TTNPB is consistent with GATA-2 up-regulation, because XLMO4 is a required coactivator of GATA-2 in ventral mesoderm (de la Calle-Mustienes et al., 2003). Neither LMO2 nor LMO4 previously has been considered an RA target. Taken together, these data suggest an important relationship among GATA, LMX1b, and LMO2/4 proteins under the control of RA signaling during hematopoiesis that remains to be explored.

Genes Involved in Steroidogenesis

XL053j05 (encoding XFFr1A), XL032j07 (encoding a gene similar to chicken CYP11A1), and XL001e06 (encoding a Xenopus homologue of LBP; Venkatesan et al., 2003) were up-regulated by TTNPB treatment. XL032j07 and XL001e06 were coexpressed in the lateral plate mesoderm (Figs. 4H, 5I). CYP11A1 transcription is known to be activated by steroidogenic factor 1 (SF1) in mouse and human (Morohashi and Omura, 1996; Gizard et al., 2002), by LRH-1 in human (Sirianni et al., 2002) and by a LBP/CP2/LSF transcription factor in human placental tissue (Huang and Miller, 2000). CYP11A1 (P450SCC, cholesterol side-chain cleavage enzyme) is the first enzyme in the synthesis of all steroid hormones. CYP11A1 mutations are found in human congenital adrenal hyperplasia, and CYP11A1 deficiency affects salt levels, sugar levels, and sexual differentiation, reflecting effects on steroid levels (Miller et al., 1989). CYP11A1 null mice could not survive after birth because of hormonal imbalance and abnormal sexual development (Hu et al., 2002). Zebrafish CYP11A1 is found in early embryos as a maternal transcript and in adult steroidogenic tissues, including brain (Hsu et al., 2002). Thus, CYP11A1 is likely to be important in Xenopus steroidogenesis. XFFr1A is Xenopus LRH-1 homolog and is similar to SF1 at the amino acid level. They belong to the Ftz-F1 nuclear receptor superfamily (NR5A) and are subcategorized to two subgroups, NR5A2 (XFFr1A and LRH-1) and NR5A1 (SF1; Commitee, 1999). Although NR5A1 is considered to be a key regulator of steroidogenesis (Achermann et al., 2001), NR5A2 is also expressed in steroidogenic tissues and activates key factors such as StAR, CYP11A1, and CYP17 (Boerboom et al., 2000; Sirianni et al., 2002). It also plays a role in bile acid biosynthesis by up-regulating the expression of enzymes such as CYP7A, the first and rate-limiting enzyme in cholesterol metabolic pathway (Nitta et al., 1999), and CYP8b1, the key enzyme for cholic acid synthesis (del Castillo-Olivares and Gil, 2000) and hepatic and pancreatic development by activation of HNF-3β, HNF-4α, and HNF-1α (Pare et al., 2001). Taken together with our results, this finding suggests that RA may have a role in steroidogenesis upstream of XFFr1A and/or XLBP by activating CYP11A1 in Xenopus embryos. The expression of CYP11A1 under the control of RA in neural tissue suggests a previously unsuspected role in neural development or patterning.

Genes Involved in Fatty Acid Metabolism

Acyl-CoA synthetases (ACSs) have a pivotal role in fatty acid metabolism by activating fatty acids to CoA derivatives before further metabolism such as β-oxidation. Two up-regulated genes, XL015a07 and XL010b24, are similar to long-chain acyl-CoA synthetase (LCS) and very-long-chain acyl-CoA synthetase (VLCS), respectively. X-linked human inherited disease adrenoleukodystrophy (ALD) is characterized by impaired peroxisomal β-oxidation associated with reduced VLCS, which causes elevated very-long-chain fatty acids (VLCFA) in tissues and blood, and the organ most affected in ALD is the brain because of high contents of VLCFA (reviewed in OMIM # 300100 and 603314 at NCBI). The Drosophila mutant bubblegum, which is a VLCS homolog, caused accumulation of VLCFA and adult neurodegeneration with marked dilation of photoreceptor axons (Min and Benzer, 1999).

The degree of sequence similarity suggests that XL010b24 may be a VLSC homolog. XL010b24 was predominantly expressed in anterior nerve system, including brain, neural crest, optic and presumptive pituitary anlagen at stage 18, and then, the expression was extended along with neural tube in addition to the anterior expression at stage 22 (Fig. 4B1–B4). The section of stage 22 embryo showed distinct staining in the roof plate of the neural tube, which is a RALDH2 domain. Although no role for VLCS has yet been documented in Xenopus development, the unique spatiotemporal expression pattern of VLCS and its up-regulation by the RAR pathway suggests a potential role in embryonic nerve system.

Expression Patterns of RAR Up-regulated Genes

We performed whole-mount in situ hybridization to determine the expression pattern of genes that were up-regulated by TTNPB and down-regulated by AGN193109 treatment. We classified the regional expression of these genes by comparison with morphological landmarks such as eye anlagen, otic vesicles, neural tube, and so on. Signature staining of RAR up-regulated genes is present in the neural tube (n = 37), the somites (n = 16), the anterior neural tissues, including the eye anlagen (n = 22), the brain (n = 9), and the neural crest (n = 13), and the mesodermal lateral plate (n = 5). Most of the genes we identified had at least regionally overlapped expression with RARα2 or RALDH2. For instance, the genes in groups 1, 2, and 3 show neural tube staining, and the genes in group 4 had lateral plate mesoderm staining. Additionally, four genes (XL010b24, XL049b08, XL010g16, and XL038g12) had staining in presumptive pituitary and eye anlagen that overlaps the anterior domain of RALDH2 expression in eye vesicles and olfactory anlagen during neurula stage (Chen et al., 2001). We found that three genes (XL002l01, XL010b24, and XL049b08) had staining in the dorsal neural tube, which was similar to the neural staining of RALDH2 in the roof plate of the rostral spinal cord observed at tail bud stage (Chen et al., 2001). On the other hand, no gene we identified showed a strict spatiotemporal coexpression with either RARα2 or RALDH2. There are at least two possibilities to explain this result. The first is that these genes might be regulated by RARβ, RARγ or some combination of the three RARs in response to treatment with TTNPB and AGN193109. The second is that these genes may be coregulated by other signals that are independent from RAR signaling, act in concert with RAR signaling, or are downstream of RAR-mediated signals. Therefore, we cannot eliminate the possibility that some of the RAR–up-regulated genes we identify here are not solely regulated by RARs. Further investigation will be required to fully understand the regulation of these genes.

The synexpression theory holds that genes with similar expression patterns are likely under the same regulatory mechanisms and that the members of synexpression groups should show tight spatiotemporal coexpression over various stages (Niehrs and Pollet, 1999). Because most of the genes we chose for expression analysis were unidentified (n = 17) or hypothetical (n = 13), we used synexpression theory to assess the potential biological roles of these unknown genes in Xenopus embryos. We divided the expression patterns of the 43 genes into five groups (Supplemental Table 5).

Overall, most of the tested genes showed distinct expression patterns, which overlapped with either RALDH2 or RARα2 expression. Many of them had discrete staining in specific tissues such as the signature staining described above and others such as the pronephros and the blood islands. Our previous study showed that zygotic expression pattern of RARα2 was higher in the spinal cord, lower in the anterior neural tube (with a sharp demarcation at the level of the hindbrain) and prominent expression in the developing eyes during neurula stage (Shiotsugu et al., 2004; see also, Sharpe, 1992). In the broad sense, expression of XL016p20 and XL043c08, exemplars for group 2 (Fig. 4C,D), was similar to RARα2 expression in the neural tube and developing eye expression with regionalized staining in the brain. Unlike RARα2, the neural tube expression did not show any strong regional demarcation. Expression of XL016p20 and XL043c08 are remarkably similar to each other at stage 18 and 22 but showed distinct differences in their response to TTNPB treatment, although they showed similar responses to AGN193109 treatment (Figs. 4S, 5U). These results suggest that these genes are dominantly regulated by the RAR signaling pathway in the posterior neural tissue but are controlled by more than one signaling pathways in the anterior tissues.

In summary, we used microarray analysis to identify genes whose expression was altered in the neurula by up-regulating or down-regulating RAR transcriptional activity. The use of improved methods, particularly Bayesian statistical analysis, allowed us to isolate RAR-responsive genes with low fold differences (1.5-fold) but high statistical significance (P < 0.01). We identified 334 up-regulated genes and 67 down-regulated genes (P < 0.01), which included known RA target genes and large number of hypothetical or unidentified genes. In addition, this analysis revealed several putative RAR target genes that have never been proposed as RA-responsive genes. Such genes include the FGF target XER81, GATA (GATA-2, GATA-4, and GATA-5b), LIM class transcription factors LOM2 and 4, XId2, NDRG1, and XFFr1A.

We determined the expression patterns of the selected hypothetical or unidentified genes at neurula stages by using whole-mount in situ hybridization. Most of the tested clones showed expression in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. The up-regulated genes were also expressed in the tissues, including notochord, somites, pronephros, and blood islands, where RA has roles in organogenesis. This analysis of the network of genes regulated by RAR signaling during early development will provide a starting point for understanding the individual pathways and how they cooperate and interact to pattern the developing embryo.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Embryo Culture and RAR Agonist/Antagonist Treatments

Xenopus eggs were fertilized in vitro as described (Koide et al., 2001) and staged according to Nieuwkoop and Faber (1967). Treatments with RAR agonists and antagonists were performed essentially as described (Blumberg et al., 1996, 1997; Koide et al., 2001). Embryos were treated in groups of 25 in 60-mm Petri dishes with 10 ml of 0.1× MBS containing 10−6 M of RAR antagonist (AGN 193109), 10−6 M RAR agonist (TTNPB), or vehicle control (0.1% v/v ethanol) from stage 7 to stage 11 at 16–19°C. For whole-mount in situ hybridization, albino embryos were prepared as described above and harvested at stages 18 and 22.

Probe Preparation, Hybridization, and Slide Scanning

Total embryo RNA was isolated using Trizol reagent (InVitrogen Life Technologies), DNAse treated and LiCl precipitated. Poly A+ RNA was isolated using OligoTex (Qiagen). The reverse transcription (RT) reaction to synthesize amine-modified DNA was performed with Superscript II enzyme, according to the manufacturer recommended protocol. Briefly, 1 μg of mRNA was primed with oligo d(T)20 in a 30-μl reaction volume containing 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.15 mM dTTP, and 0.2 mM aminoallyl-dUTP (Molecular Probes). The RT reactions were incubated at 42°C for 1.5 hr then RNA was degraded by addition of 12.9 μl of 1 M NaOH followed by incubation for 15 min at 65°C. The reaction was neutralized by the addition of 12.9 μl of 1 M HCl, and the reaction mix was diluted to 62 μl with 1 M Tris-HCl (pH 7.0). The amine-modified DNA was purified by using QIAquick PCR purification kit with the following modifications. Three washes were performed with 75% ethanol instead of the wash buffer provided in the kit. The purified DNA was eluted with 50 μl of nuclease-free water. Unincorporated nucleotides were removed by using Microcon-30 spin column filters (Amicon). DNA labeling with two reactive fluorescent dyes, Alexa Fluor 548 and Alexa Fluor 647 (Molecular Probes), was performed according to the manufacturer's protocol. Unincorporated dyes were removed using Microcon-30 spin column filters.

The probes prepared in a final volume of 18 μl were competitively hybridized to Xenopus EST microarray as described elsewhere (Shin et al., 2005). This array contains EST clones from NIBB Mochii normalized Xenopus neurula library (19,200 clones) and tail bud library (23,040 clones). Hybridized slides were scanned using GenePix 4000B scanner (Axon Instruments), and the data were saved as tiff images of both the Cy3 and Cy5 channels. Signal and background intensities were measured by GenePix Pro 3.0 and saved in an axon text file format. The entire experiment was repeated six times.

Data Analysis

No data points were eliminated by visual inspection from the initial tiff images. Instead, false spots were eliminated using the following algorithm implemented in Microsoft Excel: Algorithm = AND [OR(R2 > 0.7, F635% Sat > 0, F532% Sat > 0), (%> B635 + 1 SD) > 55, (%> B532 + 1 SD) > 55, F635-(B635 + 1 SD) >0, F532-( B532 + 1 SD) > 0], where R2 is the coefficient of determination for the current regression value, F635% Sat is the percentage of feature pixels at wavelength 635 nm that are saturated, F532% Sat is the percentage of feature pixels at wavelength 532 nm that are saturated, % > B635 + 1 SD is the percentage of feature pixels with intensities more than 1 SD above the background pixel intensity at wavelength 635 nm, % > B532 + 1 SD is the percentage of feature pixels with intensities more than 1 SD above the background pixel intensity at wavelength 532 nm, F635 is the median feature pixel intensity at wavelength 635 nm, and F532 is the median feature pixel intensity at wavelength 532 nm. The resulting data were analyzed by using Bayesian statistical analysis as implemented in Cyber-T (http://visitor.ics.uci.edu/genex/cybert/; Baldi and Long, 2001; Long et al., 2001).

DNA Sequence Analysis

Up- or down-regulated clones that did not have corresponding sequences in the XDB database (http://Xenopus.nibb.ac.jp) were PCR-amplified and sequenced using Big Dye III (Applied Biosystems). The sequences obtained were deposited into the database and compared with public databases. In addition, clones shown in microarray data sets were clustered based on sequence similarity. The resulting clusters were treated as putative unique genes in this study.

Northern Blot Analysis

Northern blot analysis was performed with 20 μg of total RNA. Probes were prepared from cDNA clones by amplification with T3 and T7 primers and directly used for in vitro transcription reactions to produce 32P-UTP–labeled sense or antisense probes using Maxiscript (Ambion).

Quantitative RT-PCR Assay

Total RNA was treated with DNase I (Ambion) to remove contaminating DNA. First-strand cDNA was generated from 1 μg of total RNA using Superscript III (Invitrogen) according to the manufacturer's recommended protocol. Gene-specific primers were designed based on sequences from public databases or generated as described above. QRT-PCR was performed with 1 μl of RT reaction in a 25-μl reaction, which contained 0.2 μM gene-specific primers and 12.5 μl of SYBR green PCR mix (Applied Biosystems) by using a DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research). All samples were analyzed in duplicate and quantitated by the comparative cycle threshold method for relative quantitation of gene expression (Livak and Schmittgen, 2001), normalized to Histone H4. Primers for QRT-PCR analysis are provided in Supplemental Table 6.

Whole-Mount In Situ Hybridization

Digoxigenin-labeled probes for whole-mount in situ hybridization were prepared by PCR amplification of specific sequences followed by transcription with T7 RNA polymerase (Megascript, Ambion) in the presence of digoxigenin-11-UTP. Whole-mount in situ hybridization was performed as described (Koide et al., 2001; Shiotsugu et al., 2004). Primers for the generation of in situ hybridization templates are provided in Supplemental Table 7.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

B.B. and K.W.Y.C. were funded by the NIH; K.A. was supported by an NRSA from the NIH; and M.M. was the supported by the UCI Minority Science Research Program.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/1058-8388/suppmat

FilenameFormatSizeDescription
jws-dvdy.20231.img.zip47916KSupporting Information file jws-dvdy.20231.img.zip
jws-dvdy.20231.tbl.doc659KSupporting Information file jws-dvdy.20231.tbl.doc

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.