Xenopus cDNA microarray identification of genes with endodermal organ expression

Authors

  • Edmond Changkyun Park,

    1. Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Republic of Korea
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  • Tadayoshi Hayata,

    1. Department of Developmental and Cell Biology, University of California, Irvine, California
    Current affiliation:
    1. Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
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  • Ken W.Y. Cho,

    1. Department of Developmental and Cell Biology, University of California, Irvine, California
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  • Jin-Kwan Han

    Corresponding author
    1. Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Republic of Korea
    • Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk, 790-784, Republic of Korea
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Abstract

The endoderm is classically defined as the innermost layer of three Metazoan germ layers. During organogenesis, the endoderm gives rise to the digestive and respiratory tracts as well as associated organs such as the liver, pancreas, and lung. At present, however, how the endoderm forms the variety of cell types of digestive and respiratory tracts as well as the budding organs is not well understood. In order to investigate the molecular basis and mechanism of organogenesis and to identify the endodermal organ-related marker genes, we carried out microarray analysis using Xenopus cDNA chips. To achieve this goal, we isolated the Xenopus gut endoderm from three different stages of Xenopus organogenesis, and separated each stage of gut endoderm into anterior and posterior regions. Competitive hybridization of cDNA between the anterior and posterior endoderm regions, to screen genes that specifically expressed in the major organs, revealed 915 candidates. We then selected 104 clones for in situ hybridization analysis. Here, we report the identification and expression patterns of the 104 Xenopus endodermal genes, which would serve as useful markers for studying endodermal organ development. Developmental Dynamics 236:1633–1649, 2007. © 2007 Wiley-Liss, Inc.

INTRODUCTION

Research interest to study vertebrate endoderm patterning and organogenesis has surged recently. Over the past years, the isolation and characterization of zebrafish and mouse mutants affecting early endoderm specification and subsequent organ formation have intensified. In addition, the molecular functions of vertebrate genes involved in endoderm formation have been extensively analyzed (reviewed in Shivdasani, 2002; Stainier, 2002; Tam et al., 2003). During vertebrate gastrulation, the three primary germ layers (the ectoderm, mesoderm, and endoderm) undergo major rearrangements and eventually endodermal cells are internalized and form gut structures. The cells of endoderm contribute to the lining of the gut, the pharynx, oesophagus, stomach, and intestine, and also to the development of organs such as the liver, gallbladder, and pancreas. It is well documented that the regional specification of gut endoderm is influenced by the signals emanating from the adjacent mesoderm (reviewed in Fukuda and Kikuchi, 2005; Horb and Slack, 2001). The process of endodermal organ formation can be simplified into four fundamental steps: formation of the endodermal precursor cells during gastrulation, morphogenesis and regionalization of the gut tube, budding of organ domains from the gut tube, and differentiation of organ-specific cell types within the growing buds (reviewed in Wells and Melton, 1999).

In the Xenopus embryo, the endoderm is formed during gastrulation when morphogenetic movements of large groups of cells results in the internalization of endodermal progenitors. Once the endoderm layer is positioned properly, the gut tube gradually becomes regionally defined along the anteroposterior and dorsoventral axes. By stage 30, the gastro-duodenal cavity has been formed, and the regional specification of the endoderm starts to take place. During this process, extensive cross-talks between the endoderm and mesoderm occur, and endodermal cells progressively commit cells to adopt a specific fate by the influence of adjacent mesoderm. By stage 39, the position of each organ has been defined, and the onset of organogenesis is revealed by swelling, budding, and coiling of specific regions of gut tube. This process is accompanied by variations in cell adhesion, mobility, attractive and repulsive cues, as well as proliferation and cell death. By stage 46, the gut endoderm has transformed from a straight tube to a complex coiled structure that is enormously elongated. The differentiation of organ-specific cell types within the growing buds commences. Although recent studies using Xenopus embryos have elucidated the molecular pathway that controls the initial formation of the endoderm germ layer (Sinner et al., 2006), how the endoderm is subsequently patterned and how highly specialized cell types of the gastrointestinal and respiratory system are generated are poorly understood. Hence, the identification of molecular mechanisms and characterization of genes that are directly involved in differentiation of the gut tube into specific tissues and organs are major issues to be resolved. To initiate such studies, we used cDNA microarrays to isolate genes that are expressed in the developing endodermal organs of Xenopus. Based on their cellular functions and expression patterns in the isolated Xenopus gut endoderm, some of these genes are expected to have crucial functions during endodermal organ development.

RESULTS

Isolation of Endodermal Organ-Related Genes Using Microarrays

In order to isolate the endodermal organ-related genes, we carried out a microarray analysis using high-density Xenopus cDNA chip (Shin et al., 2005). To this aim, we isolated the Xenopus endoderm from three different stages (St. 30, 39, and 46) and manually dissected each stage of endoderm to anterior and posterior regions. The schematic diagram of the microarray process is shown in Figure 1A. The anterior endoderm will later become major organs such as the lung, oesophagus, duodenum, stomach, pancreas, liver, and gallbladder, and the posterior endoderm will become the intestine. Two duplicate sets of three individual microarray experiments were performed using anterior endoderm cDNAs of each stage, labeled by Cy5, and the posterior endoderm cDNAs of each stage, labeled by Cy3. To increase the likelihood of identifying truly differentially expressed genes among 21,540 cDNA spots, we applied various filtering methods as described in the Experimental Procedures section. We then selected genes that are up-regulated either in the anterior endoderm or posterior endoderm at stages 30, 39, and 46, respectively. Overall, after removing redundant genes, we identified a total of 915 candidate genes that are expressed differentially between the anterior and posterior endoderm. Among these genes, 45, 143, and 136 genes are expressed in the anterior endoderm, and 116, 250, and 225 genes are expressed in the posterior endoderm at stage 30, 39, and 46, respectively (Table 1).

Figure 1.

A: Experimental approach. The Xenopus gut endoderms is isolated from three different stages of embryos during Xenopus organogenesis (St. 30, 39, and 46), and separated into anterior and posterior regions. Then, three independent microarray experiments were performed, and the transcriptional difference between anterior and posterior endoderm was analyzed. Anterior endoderm samples were labeled with Cy5 and posterior endoderm samples were labeled with Cy3, and hybridized to the same slide simultaneously. B: Illustrated anatomy of Xenopus gut endoderm. The drawings are adapted from Zorn and Mason (2001).

Table 1. Results of Competitive Hybridizationa
Approximately 16,500 genes survived
StageCriteriaNumber of genes
  • a

    Approximately 16,500 genes survived the data cleansing processes, and after removing redundant clones, 915 genes appeared to be enriched either in the anterior or posterior gut endoderm.

St. 30Anterior/posterior > 2.0-fold45
 Anterior/posterior < 0.5-fold116
St. 39Anterior/posterior > 1.7-fold143
 Anterior/posterior < 0.3-fold250
St. 46Anterior/posterior > 2.0-fold136
 Anterior/posterior < 0.5-fold225

Sequence-Based Classification of the Identified Genes

We grouped 915 candidate genes, according to the functional classification outlined in Expressed Gene Anatomy Database (EGDA, TIGR; Altmann et al., 2001), in order to achieve a general appreciation of the types of protein implicated in the organogenesis (Fig. 2). Isolated genes were categorized according to the predicted or established molecular functions and cellular processes of translated products based on the original expressed sequence tag (EST) sequences and clustering information available in XDB3 (http://Xenopus.nibb.ac.jp). As shown in Figure 2, we categorized the genes into seven groups: (1) cell signaling/cell communication (11.9%); (2) transcription factors (6.3%); (3) gene/protein expression and degradation (8.3%); (4) cell structure/motility (6.7%); (5) cell/organism defense (5.4%); (6) metabolism (20.8%); (7) unclassified/unknown function/hypothetical (12.2%). A number of genes failed to show significant homology to any known genes in databases (28.4%). A large group of genes belongs to the “cell signaling/cell communication” category. We also found a higher percentage of transcription and translation-related genes (14.6%), suggesting that induction of new genes is required for organ development. Especially, we note that transcription factors for various signal transduction pathways take part in the majority of proteins with a relatively high percentage as a single functional category. This implies that those transcription factors and their target genes induced upon upstream signaling might be involved in the endodermal organ fate specification. The genes related in the metabolism also take a relatively large portion. The reason for the no-database-matched genes forming the largest portion is unclear. While it is likely that some of these genes represent Xenopus-specific genes, some are likely to be due to the insufficient sequence information.

Figure 2.

Functional classification of clones isolated from microarray analysis. Pie chart of the classification of clones identified in the array based on EGAD categories (TIGR). Color coding for the different groupings is shown on the right.

Expression Patterns of Selected Genes in Xenopus Endoderm

From the microarray analysis, we selected, for further in situ hybridization analysis, genes that are more than 3.0-fold up-regulated in the anterior endoderm and 4.0-fold up-regulation in the posterior endoderm. In addition, the well-known transcription factors and signaling molecules that never have been reported to be expressed in the Xenopus gut endoderm were also chosen for in situ hybridization. Of the 915 identified candidate genes, the spatial expression patterns of 104 selected genes were analyzed in the various stages of dissected Xenopus endoderm. We clearly observed the distinct expression patterns relating to seven endodermal organ categories (oesophagus/stomach, lung, pancreas, liver, gallbladder, entire foregut, and intestine). We summarized the characterized cDNAs and predicted proteins based on their molecular functions and cellular processes (Table 2) and expression domains in the Xenopus gut endoderm (Figs. 3–10).

Table 2. Summary of Functional Classification and Expression Patterns of Genes Identified by Microarrays
NIBB IDProtein descriptionExpression patternFigure
OeLuPaLiGbStIn
A. Cell signaling/Cell communicaiton        
 XL060d04Lass2 [Homo sapiens] ortholog+++/− +++  Fig. 7
 XL065g01BMP receptor [Xenopus laevis]++++++ Fig. 9
 XL074p04Connexin 43.4 [Xenopus laevis]++   + n.s
 XL077n15Rab3D [Xenopus laevis]+ ++ ++++ Fig. 8
 XL077p09FrzA [Xenopus laevis]    +++ Fig. 8
 XL078a08Annexin I [Gallus gallus] ortholog    ++++/−Fig. 8
 XL078c03Follistatin [Xenopus laevis]+  +++ Fig. 9
 XL080f11Olfr1109 [Mus musculus] ortholog++      Fig. 3
 XL088h12Cbl proto-oncogene protein [Xenopus laevis]  +/−+/− +/− n.s
 XL090g16Gγ13 (Gng13) [Xenopus laevis] +   +/− Fig. 4
 XL092i08EpCAM [Xenopus laevis]+/−   ++ Fig. 8
 XL105b21Frizzled-7 [Xenopus laevis] ++   +/− Fig. 4
 XL108o12Sema6D [Mus musculus] ortholog +     Fig. 4
B. Transcription factors        
 XL056e04RFX3 [Homo sapiens] ortholog+/−    ++ Fig. 3
 XL060b05ESR-4 [Xenopus laevis] homolog+    +/− n.s
 XL064k07c-Myc II [Xenopus laevis]  +++/−++/− Fig. 8
 XL065b22USF-1 [Xenopus laevis] homolog++++++ Fig. 9
 XL074b17XBP-1 [Xenopus laevis]  ++++   Fig. 7
 XL079b04Sox2 [Xenopus laevis]++    ++ Fig. 3
 XL086e16JunD [Homo sapiens] ortholog      ++Fig. 10
 XL098c03COUP-TFII [Xenopus laevis]++   ++  Fig. 8
 XL102f24XLS13A [Xenopus laevis]   +/−++ n.s
 XL106p21ER81 [Xenopus laevis] ++     Fig. 4
 XL107g24GATA-6B [Xenopus laevis]++ +++/− ++ Fig. 5
 XL107h02Meis1 [Xenopus laevis]  +++/− ++ Fig. 5
 XL107n21Tbx2 [Xenopus laevis]   ++   Fig. 6
 XL109f09MafB [Xenopus laevis] homolog   ++   Fig. 6
 XL110i20Hairy2a [Xenopus laevis]+ +++++ Fig. 9
C. Gene/Protein expression regulation        
 XL058e09Ribosomal protein L13 [Xenopus laevis] homolog+++/−++++/−n.s
 XL059p14Id4 [Xenopus laevis]+++    +/−Fig. 4
 XL064e09BTF3 [Homo sapiens] ortholog++++++++++++ Fig. 9
 XL076d08Splicing factor arginine/serine-rich 1 (Sfrs1) [Xenopus laevis]+/−    + n.s
 XL081a04Ribosomal protein L14 [Xenopus laevis] homolog+  +++++/−n.s
 XL084k13Btbd4 [Xenopus laevis]   ++  +Fig. 7
 XL085j14HMG-17 [Xenopus laevis] homolog+++++++++++++/−Fig. 9
 XL090d02Pl10 [Xenopus laevis]++     Fig. 4
 XL090p07Lupus La protein homolog B [Xenopus laevis]  ++/− + n.s
 XL091g08RpL6 [Gallus gallus] ortholog+ ++++ Fig. 9
 XL105g24HMG-1 [Xenopus laevis]+/−+/−   +/−+/−n.s
 XL105m02HMG-14 [Xenopus laevis] homolog+++++++++++++/−Fig. 9
D. Cell structure/Motility        
 XL056d09IFT88 [Danio rerio] ortholog++   ++++ Fig. 8
 XL059n03Elfin [Homo sapiens] ortholog+   ++++ Fig. 8
 XL072g22Beta 5 tubulin [Xenopus laevis]+    +/− Fig. 3
 XL094g24COL4A5 [Xenopus laevis] homolog++  ++ Fig. 4
 XL110d16Cerebral cell adhesion molecule [Homo sapiens] ortholog +     Fig. 4
 XL110m20Claudin4L2 [Xenopus laevis] homolog  ++    Fig. 5
E. Cell/Organism defense        
 XL072k04Heat shock 70kDa protein 9B (Hspa9b) [Xenopus laevis]+++  + n.s
 XL074f10CD82 [Homo sapiens] ortholog+    + Fig. 3
 XL076p11Galectin family xgalectin-VIIa [Xenopus laevis]+/−    + n.s
 XL077n16C8β [Homo sapiens] ortholog   +/− + Fig. 3
 XL078a19Fetuin-like protein [Xenopus laevis]   ++  ++Fig. 7
 XL083h15Bf [Xenopus laevis]   ++   Fig. 6
 XL101a04T-kininogen [Rattus norvegicus] ortholog   ++   Fig. 6
 XL106c24Hsp60 [Xenopus laevis]++ ++++ Fig. 9
 XL110g20Rda288 [Xenopus laevis]+++++++ Fig. 5
 XL110o03Prefoldin subunit 5 [Homo sapiens] ortholog  +++/− +/− n.s
F. Metabolism        
 XL065p13IMPDH-II [Xenopus laevis]++      Fig. 3
 XL066f04Ecto-ATPase [Gallus gallus] ortholog   ++   Fig. 6
 XL066p20CAII [Xenopus laevis]+ ++  + Fig. 5
 XL069e05Sucrose phosphorylase [Xenopus laevis] homolog  +/−  +/− n.s
 XL072o15Hydroxysteroid 17-beta dehydrogenase 5 [Xenopus laevis] homolog+    + n.s
 XL074j16ODC 1 [Xenopus laevis]+    + Fig. 3
 XL075f24GPD2 [Xenopus laevis] homolog      ++Fig. 10
 XL076d21Cytochrome c oxidase subunit III [Xenopus laevis] homolog     + n.s
 XL082a18Maltosaccharide ABC transporter permease [Xenopus laevis] homolog+/−    + n.s
 XL085m08Sps1 [Xenopus tropicalis] ortholog+++   + Fig. 3
 XL087p03Tc1-like transposase [Rana pipiens] ortholog++ ++++ Fig. 7
 XL090f12Na+/K+ ATPase β3 [Xenopus laevis]   ++   Fig. 6
 XL091c04Mitochondrial malate dehydrogenase 2b [Xenopus laevis]++/−   ++n.s
 XL092l14SCOT [Xenopus laevis] homolog+  +   Fig. 3
 XL093l04ApoE [Xenopus laevis] homolog      ++Fig. 10
 XL095g13CarA [Aquifex aeolicus] ortholog+++  +++ Fig. 8
 XL095o2475 kDa glucose regulated protein [Homo sapiens] ortholog+    ++/−n.s
 XL096d19IleRS [Xenopus laevis] homolog ++     Fig. 4
 XL097h15Protein disulfide isomerase P5 [Xenopus laevis]  +/−+/− + n.s
 XL098l15HAI-1 [Homo sapiens] ortholog ++     Fig. 4
 XL102f19NADH dehydrogenase subunit 1 [Xenopus laevis] homolog+++++/−+/−+/−+/−n.s
 XL107g11Heparin cofactor II (serpind1) [Xenopus laevis] homolog     ++ n.s
 XL107p15Sec61β [Xenopus laevis] +++  ++ Fig. 5
 XL108e24Stard10 [Xenopus laevis] homolog+   +++ Fig. 3
 XL109f17A2M [Xenopus laevis] homolog   ++  ++Fig. 7
 XL109l03ATB0,+ [Mus musculus] ortholog ++ ++++ Fig. 9
 XL110p18PFK-M [Homo sapiens] ortholog +     Fig. 4
G. Unclassified/Unknown function/Hypothetical        
 XL087c01Golgi phosphoprotein 2 (Golgi membrane protein GP73) [Mus musculus] ortholog++ + n.s  
 XL087f18Posterior protein (Xpo) [Xenopus laevis] homolog++++++ n.s
 XL091j07G-rich sequence factor-1 [Homo sapiens] ortholog+    + n.s
 XL092l19[MGC78960] Hypothetical protein [Xenopus laevis] homolog+    +/− Fig. 3
 XL101d04[MGC52564] Hypothetical protein [Xenopus laevis]     ++ Fig. 3
 XL101p24[MGC53245] Hypothetical protein [Xenopus laevis] homolog+    ++ Fig. 3
 XL106d19Adult retina protein [Homo sapiens] ortholog+    +/− Fig. 3
 XL109m10Psoriasis-related protein [Homo sapiens] ortholog+/−    ++ Fig. 3
 XL109n06[Q7RL88] Hypothetical protein [Plasmodium yoelii] ortholog ++     Fig. 4
 XL110e12Apoptosis-related protein PNAS-4 [Homo sapiens] ortholog +   +/− Fig. 4
 XL110h22Tspan-1 [Homo sapiens] ortholog++    + Fig. 3
H. No database matched        
 XL076f19No homology++ ++++++++ Fig. 9
 XL077d08No homology+    + n.s
 XL084p12No homology++   +/− n.s
 XL091k10No homology+    + Fig. 3
 XL094o16No homology       n.s
 XL099p12No homology++++++ n.s
 XL100p12No homology + +   n.s
 XL102a02No homology  ++ + n.s
 XL105d12No homology+   ++ Fig. 3
 XL109o11No homology +   +/− Fig. 4
Figure 3.

Genes expressed in the oesophagus and stomach of Xenopus gut endoderm from stages 42–45. In situ hybridization was performed with each antisense probe. The nomenclature is base on NIBB database ID numbers.

Figure 4.

Genes expressed in the lung of Xenopus gut endoderm from stages 41–42.

Figure 5.

Genes expressed in the pancreas and other organs of Xenopus gut endoderm from stages 42–45.

Figure 6.

Genes expressed in the liver of Xenopus gut endoderm from stages 41–44.

Figure 7.

Genes expressed in the liver and other organs of Xenopus gut endoderm from stages 41–44.

Figure 8.

Genes expressed in the gall bladder and other organs of Xenopus gut endoderm from stages 42–44.

Figure 9.

Genes expressed in the foregut region of Xenopus gut endoderm from stages 41–45.

Figure 10.

Genes expressed in the intestine of Xenopus gut endoderm from stages 41–44.

Oesophagus and Stomach Expression

As the endodermal tubes form, the epithelium of the digestive tube is able to respond differently to specific mesodermal mesenchymes. This digestive tube differentiates into the oesophagus and stomach as well as the small intestine and colon. The oesophagus is that portion of the gastrointestinal (GI) tract that connects the pharynx to the stomach and is lined with a nonkeratinized stratified squamous epithelium. The walls contain either skeletal or smooth muscle, depending on the location. The upper third of the oesophagus contains skeletal muscle, the middle third contains a mixture of skeletal and smooth muscle, and the terminal portion contains only smooth muscle. The stomach is the most distensible part of GI tract. It is continuous with the oesophagus superiorly and empties into the duodenum of the small intestine inferiorly (Fukamachi and Takayama, 1980; Kedinger et al., 1990). However, the oesophagus and stomach are the least studied organs among the endoderm-derived organs.

Regulatory factor X 3 (RFX3; XL056e04), a member of the RFX family of transcription factor, is specifically expressed in the oesophagus and stomach (Fig. 3; Table 2B). In the mouse, RFX3 is also expressed in the stomach as well as the ovary, placenta, brain, lung, intestine, and testis (Reith et al., 1994). RFX3-deficient mice exhibit frequent left–right asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults (Bonnafe et al., 2004).

Inosine 5′-monophosphate dehydrogenase 2 (IMPDH-II; XL065p13) is highly expressed in the oesophagus only (Fig. 3; Table 2F). This enzyme appears to be essential to normal cell proliferation and T lymphocyte activation (Gu et al., 2000).

Beta 5 tubulin (XL072g22) is expressed in the oesophagus and also weakly localized in the stomach (Fig. 3; Table 2D). Beta 5 tubulin is a member of the globular protein tubulin that polymerizes to form microtubules. Microtubules are involved in essential functions such as cell motility as in cilia or flagella, chromosome movements, movement of organelles, and maintenance of cell shape (reviewed in Valiron et al., 2001).

CD82 (XL074f10) is expressed in both the oesophagus and stomach (Fig. 3; Table 2E). CD82 is a member of the tetraspanin superfamily/TM4SF (transmembrane-4 superfamily), which is postulated to be involved in cell morphology, motility, invasion, fusion, proliferation, differentiation, and signal transduction (reviewed in Hemler, 2005). It has been shown that several tetraspans transmembrane family members, including CD82, are expressed in the normal human epithelium from the oesophagus to colon (Okochi et al., 1999).

Orinithine decarboxylase 1 (ODC1; XL074j16) is also enriched in the oesophagus and stomach (Fig. 3; Table 2F). While ODC1 is ubiquitously expressed at early Xenopus developmental stages (until the neurula stage), the expression of ODC1 becomes localized in late stage (e.g., tadpole stage) embryos with varying intensity in specific regions, such as the branchial arch and foregut endoderm (Cao et al., 2001).

Complement component C8 beta subunit (C8β; XL077n16) expression is strongly detected in the stomach and weakly in the liver (Fig. 3; Table 2E). In the innate immune response, complement-mediated killing of pathogens through the lytic pathway is an important effector mechanism. C8 is a serum glycoprotein consisting of three subunits (α, β, and γ) encoded in separate genes, which is one of the components of the lytic pathway (Kazantzi et al., 2003).

Sox2 (XL079b04) is highly expressed in both the oesophagus and stomach (Fig. 3; Table 2B). Recently, Sox2 expression was detected in the anterior endoderm domain spanning the future oesophagus and stomach in Xenopus (Chalmers et al., 2000). This anterior domain expression, terminating at the distal end of the stomach, is identical to that reported for chicken Sox2. In chicken, the Sox2 expression appears in the rostral gut epithelium from the pharynx to the stomach before commencement of morphogenesis and cytodifferentiation, and the expression of Sox2 is regulated by the underlying mesenchyme (Ishii et al., 1998). In the mouse, Sox2 is also expressed in the developing gut endoderm (Wood and Episkopou, 1999).

Olfactory receptor Olfr1109 (XL080f11) is expressed in the oesophagus exclusively (Fig. 3; Table 1A). The olfactory receptor (OR) gene family belongs to the superfamily of seven-transmembrane-domain, G protein–coupled receptors (GPCRs) that can trigger a signaling cascade in sensory neurons. Several Xenopus ORs also have been identified and their expression patterns analyzed (Mezler et al., 1999). However, the role of OR gene family in the oesophagus is not clear.

Selenophosphate synthetase 1 (Sps1; XL085m08) is strongly expressed in the oesophagus and partially in the stomach and lung (Fig. 3; Table 2F). Sps produces the highly active Selenium donor, monoselenophosphate, and forms selenide and ATP in the Selenium metabolism process. Selenium is an essential trace metalloid, the deficiency of which can lead to both abnormal embryonic development and fertility (reviewed in Hatfield and Gladyshev, 2002).

XL091k10 has no homology match in the Xenopus database. XL091k10 is localized in the oesophagus and stomach (Fig. 3; Table 2H).

3-ketoacid CoA-transferase homolog (SCOT; XL092l14) is specifically expressed in the oesophagus and weakly in the liver (Fig. 3; Table 2F). 3-ketoacid CoA transferase, also known as succinyl-CoA:3-ketoacid CoA-transferase (SCOT), is a key mitochondrial enzyme in the metabolism of ketone bodies in various organs (Mitchell et al., 1995).

XL092l19 encodes a homolog of Xenopus hypothetical protein MGC78960. The expression of XL092l19 is detected in the oesophagus and stomach (Fig. 3; Table 2G). The putative coding region of this gene contains a WD40 domain, found in a number of eukaryotic proteins that cover a wide variety of functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing, and cytoskeleton assembly.

XL101d04 encodes a Xenopus hypothetical protein MGC52564. The expression domain of this gene in the endoderm is the stomach (Fig. 3; Table 2G).

XL101p24 also encodes a homolog of Xenopus hypothetical protein MGC53245, which is strongly expressed in both the oesophagus and stomach (Fig. 3; Table 2G).

XL105d12 does not show any homolog to the sequences in the database. The expression of this gene is detected in the oesophagus and stomach. Additionally, this gene is expressed in the anal tip (Fig. 3; Table 2H).

Xenopus ortholog of human adult retina protein (XL106d19) is enriched in the oesophagus and weakly expressed in the stomach in the Xenopus endoderm (Fig. 3; Table 2G).

StAR-related lipid transfer protein 10 homolog (StarD10; XL108e24) is expressed in the oesophagus and stomach. Additionally, this gene is specifically expressed in the gallbladder (Fig. 3; Table 2F). StarD10 contains the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain that is thought to mediate the binding of lipids, including sterols. The START proteins are suggested to function in a variety of distinct physiological processes, such as accelerating monomeric lipid exchange between the cytosolic membrane surfaces of different organelles, lipid metabolism, and modulation of signaling events (reviewed in Alpy and Tomasetto, 2005).

Psoriasis-related protein (XL109m10) is localized in the stomach at high levels and in the oesophagus at low levels (Fig. 3; Table 2G). Psoriasis is a chronic skin disease of scaling and inflammation that occurs when skin cells quickly rise from their origin below the surface of the skin and pile up on the surface before they have a chance to mature.

Tetraspanin 1 (Tspan-1; XL110h22) is expressed highly in the oesophagus and weakly in the stomach (Fig. 3; Table 2G). Tspan-1 is also a member of tetraspanins/TM4SF, like CD82. Human Tspan-1 has been identified before (Todd et al., 1998). However, the cellular and molecular function of Tspan-1 still remains elusive.

Lung Expression

The lung evolved as a system of branched conduits for air and blood coupled to a vast network of honeycomb-like alveolar structures designed for gas exchange. Primordial lung buds originate as outpouchings of the primitive foregut endoderm, and the airway tree is generated by reiterated budding and branching of these tubules. The molecular mechanisms responsible for primary lung bud induction are little understood. In the lung morphogenesis, several molecules, such as fibroblast growth factor-10 (FGF-10), sonic hedgehog (Shh), bone morphogenetic protein 4 (BMP4), and transforming growth factor β-1 (TGFβ-1), have been well studied (reviewed in Cardoso, 2001).

Inhibitor of differentiation/DNA binding 4 (Id4; XL059p14) is strongly expressed in the lung (Fig. 4; Table 2C). Id proteins dimerize with general basic helix-loop-helix (bHLH) factors as negative regulators, preventing their interaction with tissue-specific bHLH factors, to inhibit premature differentiation and to stimulate proliferation (reviewed in Yokota, 2001). In the mouse, Id1, 2, and 3 are expressed in the lung, whereas the expression of Id4 can be detected in the neuronal tissues and the ventral portion of the epithelium of the developing stomach (Jen et al., 1996).

Pl10 (XL090d02) is localized in the lung and oesophagus region (Fig. 4; Table 2C). Pl10 is one of the ATP-dependent DEAD-box RNA helicase proteins that appear to have a modulatory effect on the RNA secondary structure and thus are implicated in a range of cellular processes that involve regulation of RNA functions including RNA splicing, translation, ribosome assembly, spermatogenesis, embryogenesis, cell growth, and division (reviewed in Cordin et al., 2006).

Guanine nucleotide binding protein gamma 13 subunit (Gγ13; Gng13; XL090g16) is expressed in the lung and stomach weakly (Fig. 4; Table 2A). Heterotrimeric G proteins couple cell surface receptors for signals that regulate normal growth and differentiation to intracellular effectors. The expression of Gγ subunits is highly restricted, and the functional interaction of Gγ subunits is selective, though not limited to a single type of Gα and Gβ subunits (Schwindinger and Robishaw, 2001).

Type IV collagen alpha 5 chain homolog (COL4A5; XL094g24) is also expressed in the lung as well as the oesophagus, stomach, and gallbladder (Fig. 4; Table 2D). Type IV collagens are also called network-forming collagens since type IV molecules assemble into a felt-like sheet or meshwork that constitutes a major part of the mature basal laminae. A mutation in COL4A5, one of the type IV collagen alpha chains, caused Alprot syndrome (ATS), an inherited disorder of the basement membrane of the kidney, eye, and ear (Pescucci et al., 2003).

Isoleucyl-tRNA synthetase homolog (IleRS; XL096d19) is exclusively expressed in the lung (Fig. 4; Table 2F). IleRS is a member of aminoacyl-tRNA synthetases (ARSs), which esterify the cognate amino acids with their specific tRNAs. Although ARSs are housekeeping enzymes essential for protein synthesis, they can play non-catalytic roles in diverse biological processes. For instance, some ARSs, including IleRS, are able to form complexes with other ARSs and additional factors, such as p43, p38, and p18 (Lee et al., 2004).

Hepatocyte growth factor activator inhibitor-1 (HAI-1; XL098l15) is also strongly expressed only in the lung (Fig. 4; Table 2F). Hepatocyte growth factor (HGF) activator inhibitor-1 (HAI-1) is a type 1 integral membrane, Kunitz-type serine protease inhibitor. HAI-1 was identified as a cognate inhibitor of matriptase, HGF activator (Shimomura et al., 1997). In the mouse endoderm, the activity of HGF activator, playing an important role in the regeneration of injured gastrointestinal mucosa, is regulated by HAI-1 and -2 (Itoh and Kataoka, 2002).

Frizzled-7 (XL105b21) is highly expressed in the lung and partially in the stomach (Fig. 4; Table 2A). Frizzled-7 is a member of the Frizzled family of seven transmembrane proteins that are receptors for secreted Wnt proteins, as well as other ligands. The Frizzled family proteins are essential for embryonic development, tissue and cell polarity, formation of neural synapses, and the regulation of proliferation and many other processes in developing and adult organisms (reviewed in Huang and Klein, 2004). During the chick gut morphogenesis, noncanonical Frizzled-7 is expressed in the small intestine mesoderm (Theodosiou and Tabin, 2003). Also mouse Frizzled-7 shows high expression in crypt epithelial cells of the adult intestine (Gregorieff et al., 2005).

Ets-related 81 (ER81; XL106p21) is strongly detected in the lung (Fig. 4; Table 2B). ER81 belongs to the PEA3 subfamily of Ets (E twenty-six transformation-specific) transcription factor superfamily. Ets transcription factors play essential roles throughout development, functioning as downstream effectors of signal transduction cascades to regulate a broad spectrum of cellular processes (Oikawa and Yamada, 2003). With regard to human tissues, ER81 mRNA is highly expressed in the brain, as well as in the testis, lung, and heart (Monte et al., 1995).

Semaphorin 6D (Sema6D; XL108o12) is also localized in the lung (Fig. 4; Table 2A). Sema6D is a member of the class 6 Semaphorins, which are transmembrane proteins and contain variable, alternatively spliced cytoplasmic portions. Semaphorins are secreted, transmembrane, and GPI (glycosylphosphatidylinositol)-linked glycoproteins that regulate cell motility and attachment in axon guidance, vascular growth, immune cell regulation, and tumor progression (reviewed in Yazdani and Terman, 2006). In the mouse embryo, Sema6D is expressed mainly in the brain and lung (Taniguchi and Shimizu, 2004).

XL109n06 encodes a Xenopus ortholog of Plasmodium yoelii hypothetical protein Q7RL88. XL109n06 mRNA is highly localized in the lung only (Fig. 4; Table 2G).

XL109o11 has no homolog in the database. The expression of XL109o11 is detected strongly in the lung and very weakly in the stomach region (Fig. 4; Table 2H).

Xenopus ortholog of human cerebral cell adhesion molecule (XL110d16) is expressed in the lung (Fig. 4; Table 2D).

Xenopus ortholog of human apoptosis-related protein PNAS-4 (XL11oe12), the function of which has not been addressed, is also expressed in the lung and very weakly in the stomach (Fig. 4; Table 2G).

Muscle type Phosphofructokinase (PFK-M; XL110p18) is localized in the lung (Fig. 4; Table 2F). Phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase, EC.2.7.1.11; PFK) is a key regulatory enzyme in the glycolytic pathway, which catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate (reviewed in Nakajima et al., 2002).

Pancreas Expression

The pancreas is a soft, glandular organ that has both endocrine and exocrine functions. The endocrine function is performed by clusters of cells called the pancreatic islets, or islets of Langerhans, which further differentiates into four pancreatic endocrine cell subtypes, α, β, δ, and PP, which secrete the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide into the blood, respectively. As an exocrine gland, the pancreas secretes pancreatic juice through the pancreatic duct into the duodenum (Murtaugh and Melton, 2003). The genes regulating pancreas development have been extensively studied and this led to the identification of gene regulatory networks controlled by a number of pancreatic transcription factors that direct the development from multipotent progenitor cells to mature pancreatic cells (reviewed in Wilson et al., 2003; Jensen, 2004).

Carbonic anhydrase II (CAII; XL066p20) is strongly expressed in the pancreas and moderately in the oesophagus and stomach (Fig. 5; Table 2F). Carbonic anhydrases (CAs) are a widely expressed family of enzymes catalyzing the reversible hydration of carbon dioxide and regulating the acid-base balance in various organs. In the human pancreas, CAII has been detected only in pancreatic duct cells (Kumpulainen and Jalovaara, 1981). Recently, it has been reported that CAII expression was restricted to cells within ductal structures and glucagons-positive cells, and does not colocalize with any insulin-positive cells (Inada et al., 2006).

GATA-6B (XL107g24) reveals strong expression in the pancreas as well as in the oesophagus and stomach (Fig. 5; Table 2B). GATA-6 transcription factor is expressed in the cardiac tissue and endodermal derivatives, and is essential for the development and specific gene regulation of the heart, gastrointestinal tract and other tissues (reviewed in Maeda et al., 2005). Recently, GATA6 has been reported to maintain endodermal gene expression by TGFβ signaling in gastrulating Xenopus embryos, and has critical roles in the development of the gut and liver (Afouda et al., 2005). Moreover, GATA-6 is preferentially expressed in the developing endocrine pancreas of normal mice (Ketola et al., 2004).

Myeloid Ecotropic viral Integration Site 1 (Meis1; XL107h02) is strongly expressed in the pancreas and stomach, and weakly in the liver (Fig. 5; Table 2B). Meis1 encodes a homeobox protein belonging to the TALE (three amino acid loop extension) homeobox family that has a role in the normal development of the nervous system, and has also been implicated in tumorigenesis (reviewed in Geerts et al., 2005).

Sec61β (XL107p15) also shows pancreas and stomach expression (Fig. 5; Table 2F). The single transmembrane-spanning protein Sec61β (SecG in prokaryote) interacts with two other integral membrane proteins, Sec61α (SecY) and Sec61Γ (SecE), to form the heterotrimeric Sec61 complex. The Sec61 complex is a core component of the mammalian translocon, forming a channel across the endoplasmic reticulum (ER) membrane through which newly synthesized secretory proteins are translocated and via which membrane proteins are integrated (reviewed in Matlack et al., 1998).

Rda288 (XL110g20) is expressed through the Xenopus foregut endoderm with the highest level in the pancreas (Fig. 5; Table 2E). Rda288, also known as PAIRBP1 (Plasminogen activator inhibitor 1 RNA-binding protein or PAI-1 mRNA binding protein), is involved in mediating the antiapoptotic action of progesterone (P4) in spontaneously immortalized granulose cells (SIGCs) (Peluso et al., 2004). Its expression is also regulated by gonadotropins in granulose and luteal cells (Peluso et al., 2005).

Claudin-4-like 2 homolog (Claudin-4L2; XL110m20) is exclusively expressed in the pancreas (Fig. 5; Table 2D). Claudins, integral membrane proteins bearing four transmembrane domains, are involved in the formation of tight junctions in epithelial and endothelial cells with occludins and junctional adhesion molecules. Recently, tight junctions have been hypothesized to be involved in the regulation of proliferation, differentiation, and other cellular functions through the ability of the tight junction proteins to recruit signaling proteins (reviewed in Aijaz et al., 2006). Although Xenopus Claudins have been reported to be expressed in the endoderm from the early tailbud stage (Fujita et al., 2002) and function in determining the left-right body axis polarity (Brizuela et al., 2001), the developmental role in the endoderm remains elusive.

Liver Expression

The liver is the largest internal organ, primarily consisting of hepatocytes, and has a wider variety of functions than any other organs in the body. The hepatocyte is a polarized epithelial cell that exhibits both endocrine and exocrine properties. The biliary epithelial cells (cholangiocytes), sinusoidal endothelial cells, hepatic stellate cells (Ito cells), Kupffer cells, and pit cells (liver-specific natural killer cells) represent the majority of non-hepatocyte cell types in the liver. During embryogenesis, the liver is generated in the ventral foregut endoderm. As FGFs from precardiac mesoderm and BMPs from septum transversum mesenchyme (STM) are secreted to adjacent ventral foregut endoderm, the ventral foregut endoderm region is specified to adopt a hepatic fate. After specification, a swelling of the ventral endoderm generates the liver bud, which expresses several liver genes. Then, the pre-hepatic cells delaminate from the foregut and migrate into the septum transversum for further differentiation to functional liver. Several transcription factors have been identified, such as Hnf3/FoxA in competency, Hex in specification, and Prox1 in morphogenesis (Duncan, 2003; Zhao and Duncan, 2005).

Ectonucleoside Triphosphatase (Ecto- ATPase; XL066f04) is highly expressed in the Xenopus liver only (Fig. 6; Table 2F). Ecto-ATPase, also known as CD39L1 and NTPDase2 (ectonucleoside triphosphate diphosphohydrolase 2), is an integral membrane glycoprotein that hydrolyzes all extracellular nucleotide 5′-triphosphates. In the liver, extracelluar nucleotides regulate diverse biological functions and are important in the regulation of liver metabolism, hepatic blood flow, and bile secretion (reviewed in Schwiebert and Zsembery, 2003). It has been suggested that canalicular ecto-ATPase is involved in the efflux of bile acid in the development of the rat liver (Okada et al., 1999).

Complement factor B (Bf; XL083h15) is also exclusively expressed in the liver (Fig. 6; Table 2E). Bf is a key component of the C3 convertase of the alternative complement pathway, and its gene resides in the mammalian MHC (major histocompatibility complex) class III (reviewed in Holers, 2000).

Sodium/potassium-transporting ATPase beta-3 subunit (Na,K-ATPase β3; XL090f12) is localized in the liver (Fig. 6; Table 2F). Na,K-ATPase, a membrane-associated enzyme responsible for the ATP-dependent active transport of Na+ and K+ in most animal cells, plays an essential role in maintaining the ionic and osmotic equilibrium of the cell. In addition to pumping ions, Na,K-ATPase is engaged in the assembly of multiple protein complexes that transmit signals to different intracellular compartments (reviewed in Xie and Cai, 2003).

T-kininogen (XL101a04) mRNA is enriched in the liver (Fig. 6; Table 2E). T-kininogen is an isoforom of kininogens, the precursor proteins for small vasoactive kinins. The kininogens are synthesized in the liver and circulated in the plasma and other body fluids. In the rat plasma, T-kininogen is considered to be an acute phase reactant of inflammation. In addition, T-kininogen is suggested to be a healing protein, given its properties as a cysteine protease inhibitor and its function in interleukin 6 production (reviewed in Sharma, 2006).

T-box transcription factor 2 (Tbx2; XL107n21) also shows specific expression in the liver (Fig. 6; Table 2B). The T-box gene family is involved in early embryonic cell fate decisions, regulation of the development of extraembryonic structures, embryonic patterning, and many aspects of organogenesis. Mammalian Tbx2 is dominantly expressed in the lung and kidney, and has critical functions in the development of the heart and limbs (reviewed in Naiche et al., 2005), but their roles in the liver has not been implicated.

MafB homolog (XL109f09) transcript is localized only in the liver (Fig. 6; Table 2B). The Maf family of transcription factors has important roles in embryonic development and cellular differentiation. Mammalian MafB is reported to be mainly expressed in the lens, spleen, lung, intestine, and kidney, as well as in the liver in rats and chicken (reviewed in Reza and Yasuda, 2004).

Liver/Other Expression

Xenopus ortholog of human longevity assurance homolog 2 of yeast LAG1 (Lass2; XL060d04) is mainly expressed in the liver and oesophagus. Also, the lung and gallbladder show moderate expression (Fig. 7; Table 2A). LAG1 (longevity assurance gene 1) was originally identified in yeast Saccharomyces cerevisiae and shown to play a role in determining yeast longevity. LAG1 is localized in the ER and likely to play a role in ceramide signaling, which affects growth, proliferation, stress resistance, and apoptosis (reviewed in Jazwinski and Conzelmann, 2002). Lass2, also known as SP260 and LAG1Hs-2, is a human homolog of LAG1, and its transcript is highly expressed in the liver and kidney (Pan et al., 2001). In the mouse, TRHs (translocating chain-associating membrane [TRAM] protein homologs) have been reported to be homologs of LAG1. Especially, TRH3 is expressed in the liver as well as the kidney, lung, and intestine (Riebeling et al., 2003).

X-box binding protein 1 (XBP-1; XL074b17) is specifically expressed in both the liver and pancreas (Fig. 7; Table 2B). XBP-1 is a basic leucine zipper transcription factor in the CREB (cyclic AMP response element binding protein)/ATF (activating transcription factor) family that is involved in cell-differentiation processes. In the embryonic mouse, XBP-1 has been shown to be essential for liver development (Reimold et al., 2000).

Fetuin-like protein (XL078a19) is enriched in the liver and intestine (Fig. 7; Table 2E). Fetuin is a member of the cystatin superfamily of cysteine protease inhibitors, which encompasses a series of closely related proteins that are synthesized mostly in the liver. Fetuin has been implicated in several diverse functions, including osteogenesis and bone resorption, regulation of insulin activity and HGF activity, response to systemic inflammation, and inhibition of unwanted mineralization (reviewed in Arnaud and Kalabay, 2002).

BTB/POZ domain containing 4 (Btbd4; XL084k13) is also expressed in the liver and intestine (Fig. 7; Table 2C). The broad-complex, tramtrack (ttk) and bric-a-brac/poxvirus and zinc finger proteins (BTB/POZ) domain is highly conserved in a large family of eukaryotic proteins that is found at the N-terminus of some C2H2-type zinc finger transcription factors and in some actin-binding proteins having a kelch motif. The BTB/POZ domain appears to play diverse roles in mediating interactions among proteins that are involved in transcription regulation, chromatin structure, cytoskeleton organization, development, homeostasis, and neoplasia (reviewed in Collins et al., 2001).

Tc1-like transposase (XL087p03) is highly expressed in the liver. It also shows moderate expression in the oesophagus, lung, gallbladder, and stomach, but not in the pancreas or intestine (Fig. 7; Table 2F). The Tc1/mariner superfamily of transposable elements is one of the most diverse and widespread class II transposable elements (reviewed in Miskey et al. 2005).

Alpha 2 macroglobulin homolog (A2M; XL109f17) is dominantly expressed in the liver and intestine (Fig. 7; Table 2F). A2M is a broad-spectrum proteinase inhibitor that is abundantly present in plasma of vertebrates and invertebrates, and produced predominantly by the liver. In addition to its role as a proteinase inhibitor, A2M also functions as a binding protein to a variety of growth factors and cytokines (reviewed in Armstrong and Quigley, 1999).

Gallbladder Expression

The gallbladder is a saclike organ attached to the inferior surface of the liver. This organ stores and concentrates bile, which drains to it from the liver by way of the bile ducts, hepatic ducts, and cystic duct, respectively. Although the studies of gallbladder cancer have been continually reported on, its developmental mechanism remains largely unknown.

Intraflagella transport 88 (IFT88; XL056d09) is specifically expressed in the gallbladder, and also strongly in the oesophagus and stomach (Fig. 8; Table 2D). IFT88 (OSM-5 in C. elegans, Tg737 in mammal) is a complex B protein of IFT particle subunits that constitute IFT machinery with IFT motors and cargo molecules. IFT systems utilize microtubule-based motor proteins to move and position subcellular components, and play critical roles in organizing the cytoplasm of eukaryotic cells. In the mouse, Tg737 has been implicated in polycystic kidney disease and biliary liver cell proliferation (reviewed in Scholey, 2003).

Elfin (XL059n03) is also localized in the gallbladder, oesophagus, and stomach (Fig. 8; Table 2D). Elfin, also known as CLIM1 (C-terminal LIM domain protein 1) and CLP-36, is a member of the Enigma protein family. Enigma proteins (also named PDZ–LIM family) are a family of cytoplasmic proteins including Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP36, which contain an N-terminal PDZ domain and a series of C-terminal LIM domains. PDZ and LIM domains are protein interaction motifs that are found in various proteins associated with the cytoskeleton (reviewed in Dawid et al., 1998; Fanning and Anderson, 1999), and PDZ-LIM proteins have been suggested to act as adapters between kinases and cytoskeleton (Zhou et al., 1999; Vallenius and Mäkelä, 2002).

c-Myc II (XL064k07) shows specific expression in the gallbladder and pancreas (Fig. 8; Table 2B). The c-Myc proto-oncogene is a key regulator of cell proliferation, and its deregulated expression is detected in many tumor cell types (reviewed in Pelengaris et al., 2000), including pancreas carcinoma (Silverman et al., 1990) and gallbladder cancer (Yukawa et al., 1993). Recently, c-Myc has been reported to be expressed in human fetal and adult pancreatic tissue, but not in the differentiated endocrine cells, and has been suggested to play a role in the switch mechanism that controls the inverse relationship between proliferation and differentiation in human pancreatic endocrine cells (Demeterco et al., 2002).

Rab3D (XL077n15) is strongly expressed in the gallbladder, pancreas, oesophagus, and stomach (Fig. 8; Table 2A). Rab3D, a small Ras-like GTPase, is a key regulator of intracellular vesicle transport during exocytosis and apically directed transcytosis. Unlike other Rab3 isoforms, Rab3D is enriched in a number of non-neuronal tissues including exocrine pancreas, and localized to secretory granules in the cytoplasm of these cells (reviewed in Millar et al., 2002).

Frizzled-related protein FrzA (XL077p09) is localized in the gallbladder and stomach (Fig. 8; Table 2A). FrzA shares a sequence similarity with the extracellular Wnt binding domain of Frizzled. FrzA has been reported to play roles as a secreted antagonist of Wnt signaling, which blocks convergent extension (CE) movements and inhibits induction of secondary axes by Wnts (Xu et al., 1998).

Annexin I (XL078a08) is expressed in the gall bladder and stomach as well as weakly in the intestine (Fig. 8; Table 2A). Annexin I was first identified as a mediator of the anti-inflammatory actions of glucocorticoids in the host defence system. Annexin I exerts significant effects on several physiological and pathological processes, including cell growth, differentiation, apoptosis, membrane fusion, endocytosis, and exocytosis (reviewed in John et al., 2004).

Epithelial cell adhesion molecule (EpCAM; XL092i08) shows the gallbladder and stomach expression (Fig. 8; Table 2A). EpCAM functions in adhesion, involving homotypic calcium-independent cell–cell adhesion as one of the intercellular adhesion molecules, taking its place alongside integrins, immunoglobulin-like CAMs, selectins, and cadherins. EpCAM also has been demonstrated to function in intracellular calcium signaling under another name, tumor-associated calcium signal transducer 1 (reviewed in Armstrong and Eck, 2003). Additionally, EpCAM is reported to induce proliferation and enhance cellular metabolism along with a rapid up-regulation of the proto-oncogene c-Myc (Münz et al., 2005).

Carbamyl phosphate synthetase small chain (carA; XL095g13) is expressed in the gallbladder, oesophagus, stomach, and lung (Fig. 8; Table 2F). Carbamoyl phosphate synthetase (CPS) catalyzes the cyarbamoyl phosphate production that is employed in the urea cycle in most vertebrates (reviewed in Holden et al., 1999).

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII; XL098c03) is enriched in the gallbladder and oesophagus (Fig. 8; Table 2B). COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TFII is highly expressed in the mesenchyme of internal organs (reviewed in Pereira et al., 2000). However, a recent study has suggested that COUP-TFII is also expressed in the mouse gut endoderm (Zhang et al., 2002) and is essential for radial and anteroposterior patterning of the stomach as a target of hedgehog signaling (Takamoto et al., 2005).

Foregut Expression

General transcription factor BTF3 (BTF3; XL064e09), involved in the formation of the transcriptional complex with RNA polymerase II, is expressed in the entire foregut region (Fig. 9; Table 2C) (reviewed in Blazek et al, 2005).

Upstream stimulating factors 1 homolog (USF-1; XL065b22) is also localized in the whole foregut area (Fig. 9; Table 2B). USFs are members of the bHLH leucine zipper transcription factor family and regulate the stress and immune responses, cell cycle and proliferation, and lipid and glucid metabolism (reviewed in Corre and Galibert, 2005).

BMP receptor (XL065g01) shows foregut expression in the Xenopus endoderm (Fig. 9; Table 2A). In several organisms, BMP signaling mediates dorso-ventral patterning and anterior-posterior axis formation during embryogenesis (reviewed in Kishigami and Mishina, 2005). In the zebrafish development, BMP signaling is shown to direct the antero-posterior patterning of the endoderm (Tiso et al., 2002). In chicken, BMP is activated in all tissue layers of the GI tract during development, and is suggested to play a role in the interactions and reciprocal communications of these tissue layers (de Santa Barbara et al., 2005). Moreover, the BMP receptor conditional knockout mice study shows that BMP signaling has critical roles in endodermal morphogenesis (Davis et al., 2004).

XL076f19 has no homology hit in the Xenopus database. XL076f19 is expressed in the foregut endoderm region, except in the lung (Fig. 9; Table 2H).

Follistatin (XL078c03) mRNA is localized in the oesophagus, stomach, liver, and gallbladder (Fig. 9; Table 2A). Follistatin is one of the growing groups of proteins including noggin, gremlin, and chordin that antagonize signaling by activins and BMPs. Interestingly, in mice, follistatin enhanced the survival and expansion of pancreatic epithelial cells, but decreased the number of differentiated β-cells caused by inhibiting activin signaling (Zhang et al., 2004).

High mobility group protein HMG-17 homolog (XL085j14) is strongly expressed in the entire foregut region and weakly in the hindgut (Fig. 9; Table 2C). HMG-17, also known as HMGN2 (high mobility group nucleosome-binding domain-containing protein 2), is an abundant non-histone chromosomal protein that binds to nucleosomes and enhances transcription (reviewed in Bianchi and Agresti, 2005). HMG-17 transcript is preferentially localized to differentiating tissue regions rather than to mature structures during murine organogenesis (Lehtonen et al., 1998).

Ribosomal protein L6 (RpL6; XL091g08) is expressed in the foregut region, except in the lung (Fig. 9; Table 2C). RpL6 is also defined as TaxREB107 (Tax responsive element binding protein 107) for its binding activity to the long terminal repeats of human T-cell leukemia virus type-I. Although RpL6 has been shown to associate with FGF-2 (Shen et al., 1998), the developmental function still remains to be discovered.

High mobility group protein HMG-14 homolog (XL105m02) is strongly expressed in the entire foregut region, like HMG-17 (Fig. 9; Table 2C). HMG-14, also known as HMGN1, is a non-histone chromosomal protein that is preferentially associated with transcriptionally active chromatin. During early mouse development, HGM-14 is also suggested to modulate the cellular levels of specific proteins (e.g., N-cadherin) to affect the cellular phenotype (Rubinstein et al., 2005).

Heat shock protein 60 (Hsp60; XL106c24) shows strong expression in the oesophagus and moderate expression in the stomach, pancreas, liver, and gallbladder (Fig. 9; Table 2E). Hsp60 has primarily been known as a mitochondrial protein that serves as a molecular chaperon (reviewed in Fink, 1999). In addition, Hsp60 has been implicated in autoimmune diseases, antigen presentation, tumor immunity, and the activation and maturation of dendritic cells. Furthermore, Hsps, including Hsp60, are shown to play an important role in cell growth and apoptosis (reviewed in Wick et al., 2004).

Na+- and Cl-coupled neutral and basic amino acid transporter B0,+ (ATB0,+; XL109l03) is highly expressed in the lung and gallbladder as well as in the liver and stomach (Fig. 9; Table 2F). ATB0,+ transports both neutral and cationic amino acids, with the highest affinity for hydrophobic amino acids. ATB0,+ has been reported to be capable of transporting a broad range of zwitterionic or cationic nitric oxide synthase inhibitors (Hatanaka et al., 2001) and as a potential delivery system for a wide variety of modified drugs and prodrugs (Ganapathy and Ganapathy, 2005). Human ATB0,+ is shown to be expressed in the lung and stomach (Sloan and Mager, 1999).

Hairy2a (XL110i20) is also localized in the foregut region with the highest levels in the stomach (Fig. 9; Table 2B). Hairy genes, which encode bHLH WRPW transcriptional repressors, belong to the evolutionary conserved Hairy/Enhancer of split (HES) subclass of repressor proteins that function as effectors of the Notch signaling pathway. Hairy genes are known to play important roles in neurogenesis and myogenesis (Davis et al., 2001).

Intestine and Miscellaneous Expression

We also found three genes, Glycerol-3-phosphate dehydrogenase 2 homolog (GPD2; XL075f24), JunD (XL086e16), and Apolipoprotein E homolog (ApoE; XL093l04), localized exclusively in the hindgut region, which will later become intestine (Fig. 10).

GPD2 (Fig. 10; Table 2F) is a NAD-dependent dehydrogenase that catalyzes the reversible redox conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate.

JunD (Fig. 10; Table 2B) is a member of the Jun family of activator protein 1 (AP-1) transcription factors that is involved in fundamental biological processes such as proliferation, apoptosis, tumor angiogenesis, and hypertrophy. Previous reports have shown that JunD negatively regulates cell proliferation and is associated with cell differentiation. JunD might also participate in an anti-apoptotic pathway and act as an anti-oncogene (reviewed in Jochum et al., 2001).

ApoE (Fig. 10; Table 2F), a member of Apolipoproteins, was first described as a lipoprotein constituent of triglyceride-rich very low-density lipoprotein (VLDL) (Shore and Shore, 1973). Apolipoproteins function to mediate the binding of the lipoproteins to cell-surface receptors, act as cofactors for enzymes of lipid metabolism, and maintain the structural integrity of lipoprotein particles as they are transported. ApoE has been reported to carry out additional functions such as stimulation of cholesterol efflux from macrophages, prevention of platelet aggregation, and inhibition of proliferation of T-lymphocyte and endothelial cells. Its common structural isoforms differentially affect the risk of developing atherosclerosis and neurodegenerative disorders, including Alzheimers's disease (reviewed in Han, 2004; Greenow et al., 2005).

Besides genes that are specifically expressed in major organs, we also identified a number of genes showing various expression patterns in the Xenopus endoderm. These results are summarized in Table 2.

DISCUSSION

We successfully used a Xenpous cDNA microarray approach to identify genes that are expressed in the developing endoderm-derived organs. Of the 915 genes in the microarray, we observed the spatial expression patterns of 104 genes in the isolated Xenopus gut endoderm. Although several genes (3 genes; Sox2, Na,K-ATPase β3, and COUP-TFII) have been previously reported to be expressed in certain organs and have developmental roles, we also identified a substantial number of genes (101 genes) that have never been reported to be expressed in the gut endoderm and play a role in the organogenesis. Some of them are positively expected to have roles in the organogenesis.

In the developing Xenopus oesophagus and stomach, various types of genes are expressed. In particular, genes that are related to gene induction and morphogenesis, such as RFX3, Sox2, beta 5 tubulin, CD82, and Tspan-1, comprise a large proportion of the total number. These genes might have roles in the differentiation and morphogenetic movement of the oesophagus and stomach. In fact, mouse RFX3 has been reported to function in the establishment of left-right asymmetry of internal organs (Bonnafe et al., 2004), and thus XRFX3, which is strongly expressed in the stomach, might similarly be important for the stomach to loop in the correct direction. With regard to Sox2, its expression in chicken is regulated by the underlying mesenchyme of the gut epithelium before morphogenesis and cytodifferentiation (Ishii et al., 1998), and accordingly is implicated in regional specification of the stomach. The expressions of CD82 and C8β are also considered to have certain roles in the immune response in the stomach against pathogens derived from consumed food. Additionally, several genes, such as IMPDH-II, C8β, Sps1, SCOT, and Tspan-1, which are expressed specifically in either the oesophagus or the stomach, could be used to distinguish the boundary between those two organs.

We also found diverse genes that are expressed in the Xenopus lung. Interestingly, the molecules classified into cell signaling and gene induction categories, such as Gγ13, Frizzed-7, Sema6D, ER81, Id4, and Pl10, take up the largest proportion. These genes might be considered to have certain roles in the context of lung morphogenesis since they have similar global functions as molecules known to function in lung morphogenesis. ER81 is a target of the FGF signaling pathway in Xenopus (Münchberg and Steinbeisser, 1999). During branching morphogenesis of lung bud, local expression of FGF-10 in the mesenchyme induces chemo-attraction and epithelial growth. At the time of bud induction, proliferation is inhibited at the tips by FGF-10-mediated up-regulation of BMP4 (Lebeche et al., 1999). Therefore, we suggest that XER81 might have crucial functions during the bud formation and proliferation in the lung endoderm following initial FGF signal. XId4 may control lung morphogenesis by inhibiting premature differentiation and stimulating proliferation of progenitor cells since it inhibits the activity of all bHLH molecules and is regulated by BMP4 (Liu and Harland, 2003). In addition, the process of lung morphogenesis requires molecules that are associated with cell adhesion and motility. Accordingly, we also found genes related to adhesion and motility, such as COL4A5, HAI-1, and Sema6D.

With regard to the genes expressed in the pancreas, CAII and GATA6 showed conserved pancreatic expression like that of their mammalian orthologs. XMeis1 was previously suggested to possibly function in the transcriptional activation complex with another homeobox protein, XPbx1b, during hindbrain and neural crest development (Maeda et al., 2001; Kelly et al., 2006). Its homolog, Meis2b, also has been reported to form a trimeric complex with Pdx1 and Pbx1b, leading to cooperative activation with the pancreatic transcription factor, PTF1, which is essential for the formation of the exocrine pancreas (Liu et al., 2001). Taken together, we suggest that XMeis1 has an essential role in Xenopus pancreatic development. Additionally, the tight junction protein, Claudin-4L2, might function in endocrine cell migration and aggregation of cells within the islets of Langerhans.

With regard to liver development, we identified molecules that are potentially responsible for the liver specification. XTbx2 has been shown to be regulated by BMP4 signaling during eye morphogenesis (Sasagawa et al., 2002), and MafB expression is maintained by FGF signaling (Marin and Charnay, 2000). Because FGFs from precardiac mesoderm and BMPs from STM are required for instructing ventral foregut endoderm to become hepatic cells (Duncan, 2003; Zhao and Duncan, 2005), we suggest that Xenopus Tbx2 and MafB have roles in liver specification and bud formation. In addition, Btbd4 might be responsible for morphogenesis during liver differentiation due to its involvement within transcriptional-mediated cytoskeleton organization in other contexts. We also identified a molecule that was previously reported to be essential for liver development. In the embryonic mouse, XBP-1 has been reported to be essential for liver development (Reimold et al., 2000). Xenopus XBP-1 is also expressed in the liver and involved in BMP signaling pathway (Zhao et al., 2003). In addition, stress and immune-related molecules, such as Bf, T-kininogen, Lass2, Fetuin-like, and A2M, are likely expressed in the developing liver as well.

Among the genes expressed in the gallbladder, several molecules, such as IFT88, Elfin, and EpCAM, are associated with cell motility. Although the developmental mechanism of gallbladder formation is largely unknown, these genes might act in the morphogenesis that occurs during gallbladder formation. Interestingly, most of the gallbladder-expressing molecules that we identified are also expressed in the stomach, suggesting the possible structural similarity between the stomach and gallbladder.

In the microarray experiment, we also identified genes that are evenly expressed in the foregut region of Xenopus gut endoderm. As 8 of those 11 genes are related to signal transduction and target gene induction, we suggest that they are potentially responsible for regional specification of the foregut. Recently, XHariy2a has been shown to be regulated by Notch signaling (López et al., 2005), and provides a cell context in which a cell can interpret Notch and other extrinsic signals by controlling the responsiveness of its target genes within a negative feedback loop (Cui, 2005). As the Notch signaling pathway is required for regional specification of the pancreas in the mouse developing foregut endoderm (Fukuda et al., 2006), we suggest that XHariy2a could function as a key regulator to determine the regional identity and differentiation of Xenopus foregut endoderm. Besides genes specifically expressed in the major endodermal organs, we also identified that a number of genes involved in cell signaling and gene induction show characteristic expression patterns in the Xenopus endoderm. Even though their expression patterns look somewhat weak and diffused, these molecules might play roles in cell proliferation and differentiation during organogenesis.

In this report, we attempted to discover new markers that are useful for studying the endoderm development in Xenopus laevis. Among the 915 identified clones by microarrays, we analyzed the spatial expression patterns of 104 genes that are selected based on the gene classes and levels of expression in the isolated Xenopus gut endoderm. Based on their expression patterns and the predicated protein structures of these cDNAs, we propose that many of these genes will have roles in the endoderm development of Xenopus and other vertebrates.

EXPERIMENTAL PROCEDURES

Xenopus Embryo Manipulations

Xenopus laevis was purchased from Xenopus I, Inc. (Ann Arbor, MI). Egg were obtained from adult female Xenopus laevis primed with 800 IU of human chorionic gonadotropin (Sigma). In vitro fertilization and culture were performed as described by Sive et al. (2000) and the embryos were staged according to Nieuwkoop and Faber (1994).

Xenopus cDNA Microarray

Xenopus endoderms were isolated from three different stages during organogenesis (St. 30, 39, and 46) and separated to a anterior and posterior region. Total RNA was extracted from each endoderm region using TRIZOL reagent (Invitrogen) and further purified by LiCl precipitation. Fluorescent-labeled cDNA probes were generated by RNA amplification protocol (Wang et al., 2000). The cDNA derived from the anterior endoderm was labeled with Cy5 (red), and that derived from the posterior endoderm was labeled with Cy3 (green). Microarray experiments were performed with Set2 Xenopus cDNA microarray slides that have 21,504 clones from tailbud cDNA library as described previously (Shin et al., 2005), and the resulting data were submitted to GEO (Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/; accession no. GSE7129).

Microarray Data Analysis

GeneData Refiner® software was used to normalize the original data from GenePix Pro (GPR) files, and GeneData Expressionist® software was used to analyze the microarray data. To eliminate cDNA spots with low signal, a signal-to-noise ration of 2.5 was applied for stage 30 and 1.0 for stages 39 and 46. To select the cDNA spots that show constant values between duplicate microarray experiments, the “Filter by Variance” analysis was performed and the cDNA spots less than a variance score of 0.05 were selected. To select genes up-regulated in the Xenopus anterior endoderm, “Filter by Average Signal” analysis was used. The threshold used was 2.0-fold (stage 30 and 46) and 1.7-fold (stage 39) for anterior endoderm and 2.0-fold (stage 30 and 46) and 3.0-fold (stage 39) for posterior endoderm. To make a final list of genes, redundant genes were removed manually using contig information available on the NIBB XDB3 (http://Xenopus.nibb.ac.jp). The plasmid DNA of selected cDNA clones were obtained from the bacterial stock at −80° and verified by sequencing.

In Situ Hybridization

In situ hybridization using the digoxigenin (DIG)-UTP-labeled antisense mRNA was performed as previously described (Harland, 1991). As cDNAs were directionally cloned into pBSIISK-, library inserts were PCR amplified using T3 and T7 primers. Antisense RNA probes were generated by using a T7 RNA polymerase. The labeled probes were detected with Fab fragments from the anti-DIG antibody (Roche) conjugated with alkaline phosphatase, and visualized with the BM purple AP substrate (Roche). In situ hybridization analysis was performed using Xenopus whole embryos and gut endoderm pieces isolated from the indicated stages.

Acknowledgements

We thank Dr. Yongchol Shin and members of Dr. Ken W.Y. Cho's laboratory for their assistance for the Xenopus cDNA microarray experiment. We are also grateful to members of our laboratory for the helpful comments on the manuscript. K.W.Y.C. was funded by the NIH.