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Keywords:

  • Medaka;
  • Groucho;
  • Tle;
  • expression pattern;
  • otic development;
  • gene duplication;
  • Fugu;
  • Tetraodon;
  • zebrafish

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The highly conserved Groucho/Tle gene family has widespread functions during embryonic development and in adults. For mammalians, four full-length Tle paralogues are known, whereas the whole spectrum of this gene family in fish species has not been analysed yet. Most detailed data exist for medaka, where 3 Tle genes have been described, Tle1, Tle3, and Tle4. We now isolated 3 additional Tle genes from the medaka genome. Sequence analysis identifies these genes as Tle2a, Tle2b, and Tle3b. Database searches of genomic sequences revealed an identical set of Tle paralogues being present in distantly related fish species, indicating duplicated Tle2 and Tle3 genes for the complete teleost lineage. Like the previously analysed medaka Tle genes, the three new genes show a broad expression pattern during embryogenesis. Nevertheless, a detailed comparison of all six Tle genes reveals critical differences in certain aspects of their expression pattern. In particular, we concentrated on the activity of Tle genes during ear development and found Tle2a and Tle2b expressed in this sensory organ. Developmental Dynamics 234:143–150, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The Drosophila Groucho gene was described initially in 1968 by a viable mutation that affects the development of the Drosophila nervous system (Lindsley and Grell, 1968). After molecular characterisation (Stifani et al., 1992), homologues of the Groucho gene were isolated for C. elegans (unc37; Pflugrad et al., 1997), Xenopus (Choudhury et al., 1997; Molenaar et al., 2000), zebrafish (Wülbeck and Campos-Ortega, 1997), medaka (Lopez- Rios et al., 2003), mouse (Miyasaka et al., 1993; Leon and Lobe, 1997), rat (Schmidt and Sladek, 1993), and man (Stifani et al., 1992). Based on their high sequence conservation, they are ordered into the Groucho/Tle (Transducin-Like Enhancer of Split) gene family. Northern blot and in situ hybridisation analysis indicates that Groucho/Tle family members are widely expressed both during embryogenesis and in the adult (Stifani et al., 1992; Dehni et al., 1995; Leon and Lobe, 1997; Wülbeck and Campos-Ortega, 1997; Yao et al., 1998).

Groucho/TLE proteins do not have the ability to bind DNA on their own, but instead act as corepressors for other transcriptions factors (for review see Chen and Courey, 2000). The repressing activity depends on the DNA-binding partner and the expression level. Interactions with Groucho/TLE proteins have been demonstrated for several transcription factors (Jimenez et al., 1997; Cavallo et al., 1998; Valentine et al., 1998; Eberhard et al., 2000; Brantjes et al., 2001; Cai et al., 2003; Puelles et al., 2004). Switching of the transactivating functions towards repression is important for development of many organs such as the eye, the brain, and the kidney (Kobayashi et al., 2001; Zhu et al., 2002; Cai et al., 2003; Lopez-Rios et al., 2003). Recently, we obtained strong evidence for an important role of Groucho/TLE proteins also for otic development (Bajoghli et al., 2005). Gain-of-function experiments with a full-length Groucho protein led to an enlargement of otic vesicles, whereas dominant negative interference with Groucho function by misexpression of Amino Enhancer of split (Aes), led to hypoplasia of the vesicles. Nevertheless, the three known medaka Tle genes, Tle1, Tle3, and Tle4, are not expressed in the developing otic vesicles (Lopez-Rios et al., 2003). We, therefore, screened the medaka draft genome database for other members of the Groucho/Tle gene family and found three novel Tle members, which we then isolated from medaka embryonic cDNA.

Groucho genes are an interesting family for evolutionary studies. They share a high level of sequence conservation in amino terminal (Q-domain) and carboxyl-terminal (WD40 domain) sequences among all family members. Thus, the WD40 domain of vertebrate Tle genes shows a sequence identity of more than 86% (Stifani et al., 1992; Wülbeck and Campos-Ortega, 1997; Chen and Courey, 2000). Furthermore, whereas a single Groucho gene was identified in Drosophila, four TLE homologues are present in higher vertebrates such as humans and mouse. Generally, gene and genome duplication have been described as the driving force for evolution (Ohno, 1970). Genome duplications generate paralogous groups of redundant genes in a species, offering the potential for the development of new functions. Whereas protostomes, such as Drosophila and deuterostomic ancestors of vertebrates tend to have single copies of genes, chordate genomes typically have more copies of a gene, often four (Holland and Garcia-Fernandez, 1996; Spring, 1997). Based on these data, it was proposed that the vertebrate genome underwent two rounds of duplication during evolution (Ohno, 1970; Spring, 1997; Skrabanek and Wolfe, 1998).

Here we report the isolation and characterization of three new medaka Tle genes. Comparison of the predicted amino acid sequences indicates high structural homology to the other members of the Groucho/TLE protein family in vertebrates and allowed us to identify the new genes as Tle2a, Tle2b, and Tle3b. Analysis of the genomic organisation of the Groucho/Tle family provides evidence for both gene- and genome duplication events during teleost evolution. Whole-mount in situ hybridisation experiments demonstrate widespread but differential expression patterns for all Tle family members.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Identification of New Medaka Groucho/Tle Genes

In a BLAST search with the amino acid sequences of human TLE genes against the medaka draft genome database (UT Genome Browser, http://medaka.utgenome.org), we found three genes positioned on the scaffolds 8464, 12474, and 199. The sequences show high similarity to the Groucho/Tle family, but are distinct from the three known Tle genes in medaka (Tle1, Tle3, and Tle4; Lopez-Rios et al., 2003). Partial coding sequences of these genes were amplified by RT-PCR from 3-day-old embryos and verified by sequencing.

Outside the isolated cDNA fragments, we had to rely on automatically performed predictions of exon/intron structure. We first concentrated on the highly conserved WD-40 domain, where the predicted sequences were of good quality (60 amino acids of the last 2 exons were omitted due to problems of one predicted sequence). Initially, we defined a consensus sequence for the known human, mouse, rat, chick, Xenopus, zebrafish, and medaka Groucho/Tle genes (Fig. 1A). We only considered full-length family members classified into the paralogous groups Tle1 to 4. Alignment of these sequences revealed characteristic amino acids unequivocally identifying the four Tle genes of vertebrates (see dark shaded positions in Fig. 1). Comparison of the sequences at the characteristic positions in the WD40 domain revealed a 100% match of the new medaka genes with either the Tle2 class (2 sequences) or the Tle3 class (1 sequence). Based on these results, we named the new genes Tle2a and Tle2b and Tle3b (in order to differentiate it from the previously published Tle3, which we named here Tle3a).

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Figure 1. Classification of new medaka Tle genes. A: The C-terminal amino acid sequences of all known full-length vertebrate Groucho/Tle genes (human, mouse, rat, chick, Xenopus, and zebrafish sequences corresponding to human TLE1 from amino acid 420 to 710; for accession numbers see the Experimental Procedures section) were aligned and a consensus sequence determined. Alignments for the individual classes (TLE1 to 4) were used to detect characteristic amino acids, exclusively identified in individual classes (marked with black overlay). Sequences of the new medaka Tle genes were then compared and the characteristic amino acids (marked with grey overlay) used to assign these genes to the different Tle classes. Dots indicate identical amino acids compared to the consensus sequence, asterisks are used for variable positions. Dashes indicate gaps in the alignment. The four WD dipeptides are boxed. B: Unrooted tree established from an alignment of amino acid sequences of Groucho/Tle proteins corresponding to human Tle1 from position 145 to 735. Tetraodon (Tn), zebrafish (2f), and medaka (Ol) sequences were obtained using the Genewise program.

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Tle2 and Tle3 Genes Are Duplicated in Teleost Fish

Having identified 6 full-length Tle family members in medaka, we next asked the question whether a similar spectrum of Tle genes exists in other fish species. We, therefore, searched for paralogous TLE genes in databases of the teleost fish Tetraodon (Tetraodon nigrovirdis), Fugu (Fugu rubripes), and zebrafish (Danio rerio). Phylogenetic studies place medaka close to Fugu and Tetraodon species, with an estimated divergence time of 60–80 million years (Myr) ago (Powers, 1991), which is less than the evolutionary distance between man and mouse. Zebrafish is more distantly related to medaka. It is estimated that these two fish separated from their last common ancestor ∼180 Myr ago (Powers, 1991). By using BLAST with human TLE sequences, we found six Tle genes both in the Tetraodon and the Fugu genome database (http://ensemble.org). Analysis of the predicted sequences of both species based on the characteristic amino acids within the WD-40 domain gave rise to exactly the same composition of Tle paralogues as for medaka (data not shown, for accession numbers of the identified genes see the Experimental Procedures section). In zebrafish, two Tle genes have been isolated (Gro1 and Gro2; Wülbeck and Campos-Ortega, 1997). Both are considered as homologues of Tle3, which is consistent with our alignments in the WD-40 domain (data not shown). In addition, we identified two full-length Tle2 homologues in the zebrafish draft genome database (http://ensemble.org). Surprisingly, no Tle1 or Tle4 gene appeared in these searches, which might be due to the draft character of the sequence database.

In order to verify the assignment of all fish Tle genes into the individual classes, we performed sequence comparisons of the full-length amino acid sequences. Nevertheless, these sequences were of poor quality, due to several misinterpretations of the automatically working annotator, which resulted in inconsistent alignments. We, therefore, applied the Genewise program (Birney et al., 2004) for a complete new sequence prediction. Using the human TLE1 amino acid sequence as a reference, we indeed obtained high-quality sequence predictions for all fish Tle genes. A pylogenetic tree deducted from these sequences (Fig. 1B) fully supports our analysis of the WD40 domain. Taken together, both overall sequence composition as well as individual characteristic amino acids identify duplicated Tle2 and Tle3 orthologues in distantly related teleost fish species.

Evidence for Both Gene and Genome Duplication Events During Evolution of Tle Genes in Teleost Fish

Two rounds of whole genome duplications during vertebrate evolution are the most likely mechanism explaining the presence of up to four paralogues for each gene. Thus, four Hox clusters are found in Sarcopterygii (a monophyletic group including lobe-finned fish, amphibians, reptiles, and mammalians), whereas a single one is typically found in non-vertebrates (Amores et al., 1998; Aparicio, 2000). Medaka, zebrafish, Fugu, and Tetraodon possess more than four Hox clusters, suggesting additional duplications in teleost fish (Postlethwait et al., 1998; Prince et al., 1998; Meyer and Schartl, 1999; Jaillon et al., 2004). Recently, a detailed comparison of the Tetraodon and the human genome identified large syntenic regions and thus gave strong evidence for a third entire genome duplication event during evolution of actinopterygian fish (Jaillon et al., 2004).

Based on these results, we determined the chromosomal localisation of the duplicated Tle genes. Due to its high quality, we selected the Tetraodon sequence database for this analysis. We found Tle3a and Tle3b localized on separate chromosomes (13 and 5, respectively). As expected for a genome duplication event causing the presence of two Tle3 genes, about 50% of Tetraodon chromosome 5 is syntenic to chromosome 13 (Jaillon et al., 2004). Furthermore, the human TLE3 gene is positioned on chromosome 15, which has the largest syntenic regions on Tetraodon chromosome 5 and 13 (Jaillon et al., 2004). To get more detailed information, we analysed the genes in the proximity of the two Tetraodon Tle3 genes. Indeed, for both Tle3 genes we could identify homologous genes being present at the same position flanking the human TLE3 gene (Fig. 2A), thus giving strong evidence for syntenic regions and, therefore, a genome duplication event giving rise to the two Tle3 genes in fish.

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Figure 2. Chromosomal organisation of the duplicated Tle2 and Tle3 genes. A: Schematic view of chromosomal regions containing the Tle3 locus in humans and Tetraodon. The human TLE3 gene localizes to chromosome 15, Tetraodon Tle3a, and Tle3b to chromosomes 13 and 5. Neighbouring genes being homologous are marked identically. B: Similar schematic view of the Tle2 loci. Human TLE2 localizes to chromosome 19, in Tetraodon Tle2a and Tle2b are positioned directly adjacent to each other in opposite orientation on chromosome 1. Again flanking homologous genes indicate syntenic chromosomal regions. Ensemble accession numbers for the Tetraodon genes are given above the boxes (GSTENT000 has to be added before each number); those for Tle2a, Tle2b, Tle3a, Tle3b are GSTENT00028785001, GSTENT00028784001, GSTENT00016046001, and GSTENT00018114001, respectively. Map positions in Mb are given below the boxes.

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The two Tle2 paralogues were analysed the same way. Interestingly, in the Tetraodon genome these two genes are positioned directly adjacent to each other on chromosome 1 in opposite orientation with a distance of ∼4 Kb (Fig. 2B). Tetraodon chromosome 1 has syntenic regions with human chromosome 19 (Jaillon et al., 2004). Indeed, the human TLE2 gene is positioned on this chromosome and flanking homologous genes are found on the human and the Tetraodon chromosome (Fig. 2B). This arrangement of the two Tetraodon Tle2 genes is typical for a local gene duplication. To see whether such an event might have occurred specifically in the Tetraodon lineage, we analysed the chromosomal arrangement of the two Tle2 genes in other fish species. Due to limits of the databases, we had to restrict this analysis to zebrafish, where we found both genes positioned on chromosome 22, close to each other (at the positions 18.7 and 19.3 Mbp in opposite orientation). The same tandem arrangement in a distantly related fish species suggests an ancient gene duplication event for the Tle2 gene, which might even predate the separation of the teleost lineage.

Interestingly, close to the human TLE2 gene on chromosome 19, an additional gene with Tle sequence homology has been identified (TLE6; see Fig. 2B). This gene lacks major parts of the inner domains (GP, CcN, and SP) and shows considerable deviations in the N-terminal Q-domain and the C-terminal WD-40 domain when compared to other full-length Tle members (the sequence identity in the WD-40 domain, which is higher than 86% for all full-length vertebrate Tle genes, drops to 30% for TLE6). A possible explanation for the presence of this additional Tle-like gene would be a localised duplication of the Tle2 gene in the mammalian lineage. Another hypothesis would be that the presence of two adjacent Tle2 genes was the ancient situation for vertebrates. This arrangement was kept in the fish lineage, but might have resulted in the Tle6 and Tle2 genes of mammals. Indeed, the highly aberrant Tle6 genes are positioned within the Tle2 branch of the phylogenetic tree (Fig. 1B).

All Tle Genes Are Expressed in the Central Nervous System

The expression of the three novel Tle paralogues, Tle2a, Tle2b, and Tle3b, was analysed by whole-mount in situ hybridization in medaka embryos. In agreement with other Tle genes, all transcripts show a diffuse pattern, making clear demarcations of expression domains difficult (Lopez-Rios et al., 2003; Jochen Wittbrodt, personal communication).

Tle2a transcripts are first detected in the tail bud at stage 20 (4 somites, Fig. 3A). Expression in the diencephalon and the mesencephalon starts at stage 25 (19 somites), with particular high levels in the mid-hindbrain boundary region (Fig. 3C,D).

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Figure 3. Embryonic expression of Tle2a. In situ hybridisation experiments of medaka embryos with a probe against Tle2a. Dorsal views of embryos at stage 20 (A), stage 25 (C), and lateral views at stage 23 (B) and stage 26 (D). Anterior is to the left, except for E and F, which show transverse sections of otic vesicles (dorsal to top). A: Expression of Tle2a is initiated at stage 20 in the tail bud region (arrow), The intensity of expression increases up to stage 23 (B, arrow and D). C,D: Starting from stage 25, Tle2a transcripts appear in the central nervous system, in the diencephalon, mesencephalon, optic tectum, and midbrain-hindbrain boundary (arrow). At stage 26, Tle2a expression was detected in otic vesicles, mainly at the lateral crista (arrows in F), but not during earlier stages (stage 24; E). di, diencephalon; e, eye; ev, eye vesicle; l, lens placode; mhb, midbrain-hindbrain boundary; ot, optic tectum; ov, otic vesicle; tb, tail bud; t, tail; tel, telencephalon. Scale bars = 100 μm for A, 200 μm for B, 120 μm for C, 250 μm for D, 25 μm for E and F.

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In contrast to its paralogue Tle2a, Tle2b is broadly expressed in the embryonic body during late gastrulation (Fig. 4A). At the two-somite stage (stage 19), the expression concentrates in the prospective mid-hindbrain region (Fig. 4B). Tle2b is also expressed in the tail bud, but whereas the intensity of expression for Tle2a increases (Fig. 3B,D), Tle2b transcripts disappear in this region with time (Fig. 4C). During later stages (starting at stage 25; 19 somites), Tle2b transcripts were detected in more anterior structures of the embryo, from the rhombencephalon to the telencephalon including the mid-hindbrain boundary region (Fig. 4C,D).

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Figure 4. Embryonic expression of Tle2b. In situ hybridisation experiments of medaka embryos with a probe against Tle2b. Dorsal views of embryos at stage 17 (A), stage 19 (B), stage 25 (D), stage 27 (F), and lateral views at stage 26 (C). Anterior is to the top (A,D,F) or to the left (B,C). E: Histological section at the level of the eyes (dorsal to top, lateral to left, position of section indicated by a line in D). A: Tle2b is expressed during late gastrulation in the embryonic body including the presumptive regions of otic and optic vesicles. B: At stage 19, the expression concentrates in the prospective midbrain-hindbrain region, the prospective otic vesicle region (arrow), the eye vesicles, and the tail bud. During somitogenesis expression of Tle2b is shifted to the anterior part of the embryo: the optic cup (C), lens placode (C, E), diencephalon (C–E), midbrain-hindbrain boundary (C,D), pectoral fin (F, the dotted line demarcates the pectoral fin bud), and otic vesicle (C,G). di, diencephalon; ev, eye vesicle; lp, lens placode; mhb, midbrain-hindbrain boundary; oc, optic cup; ov, otic vesicle; pf, pectoral fin; pov, prospective otic placode region; tb, tail bud; tel, telencephalon. Scale bars = 75 μm for A; 50 μm for B, 100 μm for C–E, 60 μm for F, 30 μm for G.

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Like Tle2b, Tle3b expression is first detectable during late gastrulation in the forming embryonic body (Fig. 5A), later concentrating in the prospective mid-hindbrain region (two-somite stage), with peak values observed at the boundary region (Fig. 5B,E). At stage 25, the expression in the hindbrain disappears, remaining in the di-, mes-, and metencephalon (19 somites; Fig. 5C,F). Both Tle3b and Tle2b are expressed in the pectoral fin buds at stage 27 (Figs. 4F, 5F).

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Figure 5. Embryonic expression of Tle3b. In situ hybridisation experiments of medaka embryos with a probe against Tle3b. Dorsal views of embryos at stage 17 (A), stage 19 (B), stage 21 (E), stage 25 (C), and a lateral view at stage 27 (F). Anterior is to the left, except for A where it is to the top. D: Transverse section of the eye at stage 24 (lateral to the top). Tle3b expression is detectable during late gastrulation in the embryonic body (A) and during early somitogenesis is mainly seen in the prospective midbrain-hindbrain region (B,E). At later stages, Tle3b transcripts in the CNS become restricted to the midbrain and midbrain-hindbrain boundary (C,F). During eye development, the initial broad expression becomes first restricted to the prospective neural retina (E, arrow; the dotted line demarcates the optic vesicle) and is subsequently lost (D,F). The dark colour in D represents pigmented epithelium; no transcripts could be detected in the eye at stage 24. Tle3b is also expressed in the pectoral fin (F, arrow). di, diencephalon; ev, eye vesicle; fb, forebrain; l, lens placode; mb, midbrain; mhb, midbrain-hindbrain boundary; oc, optic cup; ov, otic vesicle; pf, pectoral fin; tb, tail bud; tel, telencephalon. Scale bars = 100 μm for A,C; 120 μm for B,E, 75 μm for D, and 200 μm for F.

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Expression of Tle Genes in Sensory Organs

Based on their well-established function in sensory organ formation, we compared the expression of all Tle members in these structures and first concentrated on ear development. To determine expression of Tle genes during preplacodal stages, we marked the region with a differentially labelled probe against Pax2, which is expressed in preotic cells (Pfeffer et al., 1998). Tle1, Tle3a, Tle3b, and Tle4 do not show any expression in the domain marked by Pax2 (data not shown) and also not during later stages of otic development (Fig. 6A,D–F), although Tle1 displays strong expression in surrounding cells of the ear in the hindbrain region (Fig. 6A). However, we detected specific expression of Aes, Tle2a, and Tle2b in this organ at different stages of development. Tle2a transcripts are not present at preplacodal- and otic placode stages (Fig. 3A,B). Weak expression in otic vesicles is first seen at stage 26 (22 somites; Figs. 3D,F and 6B) and then becomes prominent in the adjacent ventral epithelial region (Fig. 3F). Tle2b is strongly expressed in the presumptive otic cells throughout late gastrulation (Fig. 4A). This expression is still detectable in the preplacodal region at stage 19 (Fig. 4B) and remains mainly in the medial region of the otic vesicle, more restricted to the prospective anterior macula (Figs. 4G, 6C). Aes is located in a band of epithelilium in the medial region of the otic vesicle at stage 24 (Fig. 6G).

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Figure 6. Expression of medaka Tle genes in the otic vesicle. In situ hybridisation experiments of medaka embryos with probes against all Tle genes. Lateral views of embryos at stages 24–26 (anterior is to the left and dorsal to the top). Only Tle2a, Tle2b, and Aes are expressed in otic vesicles (B,C,G). A:Tle1 is not expressed in the otic vesicle (the dotted line demarcates the otic vesicle), but is strongly expressed in surrounding cells of the hindbrain (asterisk). Tle3a, Tle3b, and Tle4 transcripts could also not be detected in otic vesicles (D,E,F). H: Sense control probe for Tle2b. a, anterior; SC, sense control; v, ventral. Scale bars = 25 μm for A,D,E–H and 20 μm for B,C.

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Previous studies of the eye domain have shown expression of Tle1 in the optic cup and Tle3a in the lens (Lopez-Rios et al., 2003). In contrast, we detected Tle3b mRNA only in the prospective neural retina up to stage 21 (Fig. 5E), but not at later stages (Fig. 5D,F) and also not in the lens (Fig. 5C,D,F). Whereas Tle2a transcripts were not detected in the developing eye (Fig. 3A–C), Tle2b shows expression at high levels throughout the optic vesicle (Fig. 4B), optic cup (Fig. 4D,E), and the lens (Fig. 4C,E).

Even though different Groucho/Tle genes are expressed in olfactory placodes of mouse, zebrafish, and Xenopus (Dehni et al., 1995; Leon and Lobe, 1997; Molenaar et al., 2000), unexpectedly we did not detect expression of any Tle family members in the nasal placode of medaka embryos (Table 1).

Table 1. Expression of Mcdaka Tle Genes During Embryogenesisa
 Tle1Tle2aTle2bTle3aTle3bTle4Aes
  • a

    Expression of all 6 full-length Tle genes and Aes was analysed by in situ hybridisation experiments of medaka embryos between stage 19 (2 somites) and 28 (30 somites). (+) indicates expression. (−) indicates no expression during any of the observed stages.

Telencephalon+++
Nasal placode
Optic stalk++
Optic placode++++
Optic vesicle++++
Optic cup++++
Lens placode+++
Midbrain++++++
MHB++++
Hindbrain++++
Otic placode++
Otic vesicle+++
Pectoral fin++
Spinal chord++
Somites+
Tail bud++++

Differential Expression of Tle Genes During Embryonic Development

When we compared the expression patterns of all 6 Tle genes, it became clear that considerable overlap exists. During early stages of development, broad expression was observed for most Tle genes, in particular in the CNS and sensory organs. However, significant differences between all Tle members exist and are summarized in Table 1. The close proximity of the 2 Tle2 loci also affects their regulation. The expression domains of the two genes largely overlap, although Tle2a is activated much later in a restricted pattern compared to Tle2b. On the contrary, the expression of Tle3a and Tle3b differs considerably, suggesting independent function and regulation of the two separated Tle3 genes.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Isolation of Medaka Tle2a, Tle2b, and Tle3b

Total RNA was extracted from 2–3-day-old medaka embryos using the Roti-Quick-Kit (ROTH). Reverse transcription was done with 1 μg total RNA using Revert Aid M-MuLV Reverse Transcriptase (Fermentas) and random primers. The primers used for the PCR were as follows: forward primer for Tle2a, 5′-GCGAATTCA- GCGCCATCTGTGCTCAG-3′; reverse primer for Tle2a, 5′-GCGGATCCATGGGACACCACGCCAAA-3′; forward primer for Tle2b, 5′-GCGAATTCACCAAGCGGGCCTCCACAAT-3′; reverse primer for Tle2b, 5′-GCGGATCCAGCCAGCCACTCGCCTGTC-3′; forward primer for Tle3b, 5′-GCGAATTCCCAATCTGCCTCTGACT-3′; reverse primer for Tle3b, 5′-GCGGATCCAGGGACCCGTTCATCTCAT-3′. PCR conditions were as follows: denaturation at 95°C for 10 min, then 35 cycles at 94°C for 20 s, annealing (for Tle2a at 67°C, for Tle2b at 70°C, and for Tle3b at 52°C) for 30 s and 72°C for 1 min. The DNA fragments were isolated, subcloned into the pGEM-T easy vector (Promega, Madison, WI), and sequenced.

Sequence and Genomic Analysis

For identification of Groucho/Tle genes in Fugu (Fugu rubripes), Tetraodon (Tetraodon nigrovirdis), and zebrafish (Danio rerio), we used the BLAST search program available at the ensemble genome database site (http://www.ensemble.org) in the versions: Sept 04 (zebrafish), v2.0 May 04 (Fugu), and v7 Sept 04 (Tetraodon). Scaffold numbers for the identified Tle genes in the Fugu genomic sequence database are given in brackets: Tle1 (565), Tle2a (270), Tle2b (5757), Tle3a (427), Tle3b (1184), and Tle4 (801). In the Tetraodon database Tle1 (SCAF 7076), Tel2a and Tl2b (SCAF 14995), Tle3a (SCAF 14555), Tle3b (SCAF 14581), and Tle4 (14999) were identified. Other vertebrate Groucho/Tle sequences were obtained from the NCBI server under the following accession numbers: mouse Tle1, NM_011599; mouse Tle2, NM_019725; mouse Tle3, BC056465; mouse Tle4, NM_011600; rat Tle3, NM_053400; rat Tle4, NM_019141; chick Tle1, XM_425024; chick Tle4, AB080584; Xenopus Tle4, AJ224945; human TLE1, NM_005077; human TLE2, NM_003260; human TLE3, NM_005078; human TLE4, BC059405; zebrafish Gro1, Y12467; zebrafish Gro2, NM_131012; medaka Tle1, AY158892; medaka Tle3, AY158893 and medaka Tle4, AY158894.

Full-length sequences for phylogenetic analysis were obtained using the Genewise program (http://www.ebi.ac.uk/Wise2/). ClustalW (http://www.ebi.ac.uk) was used for alignment and a tree was plotted using DrawTree software (http://www.bioasp.nl).

In Situ Hybridization and Histological Analysis

Medaka embryos of the Cab inbred strain were used for all experiments. Embryonic stages were determined according to Iwamatsu (2004). Embryos were fixed over night in 4% paraformaldehyde/2xPTW (PBS/Tween) at 4°C and subsequently dechorionated manually. Whole-mount in situ hybridization was performed as described (Quiring et al., 2004) using DIG- or FITC-labelled RNA probes.

For histological sections, after the in situ hybridisation procedure embryos were washed with PTW several times and fixed in 4% PFA/PTW for 4 hr at room temperature under continuous shaking. The embryos were then gradually dehydrated with ethanol. For sectioning, they were incubated in xylol two times for 10 min and embedded in paraffin at 55 embryos C for 20 min. Sections of 10 μm were made using a microtome.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We thank Christina Murko for help with in situ hybridisation experiments, Daniele Soroldoni for sequencing, and Magdalena Helmreich for excellent technical support. We are grateful to Javier Lopez-Rios for medaka Tle1, Tle3, and Tle4 probes. Instrumental to this work were medaka genomic sequences freely provided by the National Institute of Genetics and the University of Tokyo for use in this publication only.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
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