genes in neurogenesis and eye development - NCBI

The EMBO Journal vol. 12 no.4 pp. 1403 - 1414, 1993

Functional conservation of vertebrate seven-up related genes in neurogenesis and eye development

Anders Fjose"'3, Svanhild Nornes1, Ursula Weber2 and Marek MIodzik2 1Department of Molecular Genetics, Institute of Medical Biology, University of Troms0, N-9000 Troms0, Norway and 2Differentiation Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, Germany 3Present address: Department of Biochemistry, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway Communicated by M.Noll

Several members of the steroid receptor superfamily, including the transcription factor COUP, are closely related to the Drosophila gene seven-up (svp) which is required for the development of the embryonic central nervous system (CNS) and specific photoreceptor cells of the eye. We have identified and characterized two zebrafish (Brachydanio rerio) members of this subfamily of orphan nuclear receptors. While one of them (svp[44]) is the zebrafish cognate of COUP, the second (svp[46]) seems to be a novel member of the COUP/svp group. The proteins encoded by both genes contain highly conserved DNA-binding and putative ligand-binding domains, indicating close similarities in target sequence recognition and ligand binding. Analysis of the spatial distribution of their transcripts in whole-mount embryos revealed that the CNS is a major site of expression for both genes. At early embryonic stages, both genes are expressed in domains corresponding to specific rhombomere primordia in the hindbrain. This suggests an involvement in hindbrain segmentation and/or rhombomere specification. Moreover, transcripts derived from both genes are detected within distinct areas of the eye rudiments, suggesting roles in eye patterning and/or cell differentiation. In the case of the svp[44] gene, expression is also observed within specific parts of the midbrain, diencephalon and telencephalon. These results represent the first evidence that at least some of the nervous system and eye-specific functions of Drosophila svp are conserved in vertebrates. Key words: COUP/eye development/hindbrain segmentation/ nuclear receptor/regionalization

Introduction In both Drosophila and vertebrates the majority of developmental regulatory genes identified belong to a few evolutionarily conserved gene families such as Wnt, Par and several subgroups of homeobox genes (reviewed by McMahon, 1992; Gruss and Walther, 1992; McGinnis and Krumlauf, 1992). Within these families several vertebrate genes are thought to be structural homologues of specific Drosophila genes. However, for the various homologous pairs the levels of structural and functional conservation are (©

Oxford University Press

quite variable. Transgenic analyses of several mammalian homologues of the Drosophila homeotic genes have provided direct evidence for conservation of developmental regulatory functions, despite the fact that the structural conservation between the corresponding proteins is relatively low (McGinnis et al., 1990; Malicki et al., 1992; McGinnis and Krumlauf, 1992). In the case of the steroid receptor superfamily, considerably less is known about the functional conservation between the vertebrate and Drosophila members, since very few structural homologues have been identified (Segraves, 1991). Nevertheless, the proteins derived from the 32 genes which are known for this superfamily have common features in terms of the organization of functional domains (Evans, 1988; Green and Chambon, 1988; Beato, 1989). These nuclear receptors have DNA-binding domains with zinc finger motifs and the majority also have related dimerization, transactivation and ligand-binding domains in the same relative positions (Green and Chambon, 1988; Beato, 1989; Power et al., 1992). Despite extensive biochemical and structural analyses of DNA- and ligand-binding properties of several of these receptors (Evans, 1988; Green and Chambon, 1988; Power et al., 1992; Tran et al., 1992), relatively little is known about their developmental functions. However, recent investigations on retinoids and their corresponding receptors in vertebrate embryos have revealed evidence that both retinoic acid receptors (RAR) and retinoid X receptors (RXR) may play important roles in several aspects of development including limb patterning, specification of the body axis and neural development (Dolle et al., 1989; Eichele, 1989; Kessel and Gruss, 1991; Ruberte et al., 1991; Mangelsdorf et al., 1992). It is still unclear how specific retinoid response programmes are restricted to certain tissues and cell types at the level of transcription. Nevertheless, it seems that the formation of RAR/RXR heterodimers is an important feature of this regulation (Yu et al., 1991; Bugge et al., 1992; Kliewer et al., 1992a). In Drosophila, analyses of the mutant phenotypes of knirps (kni), tailless (tll), ultraspiracle (usp) and seven-up (svp) have revealed regulatory functions associated with anteroposterior axis specification, segmentation and neurogenesis (Niisslein-Volhard and Wieschaus, 1980; Mlodzik et al., 1990; Pignoni et al., 1990; Oro et al., 1992). Interestingly, both usp and svp, which are structural homologues of the vertebrate RXR and COUP/ARP-1 proteins respectively, are required for eye development or differentiation (Mlodzik et al., 1990; Oro et al., 1992). Another common feature of these two Drosophila proteins is that their ligand(s) have not been identified. Nevertheless, Usp shares a relatively high degree of sequence identity within its ligand-binding domain with RXR (49%), the 9-cis-retinoic acid receptor (Oro et al., 1990; Mangelsdorf et al., 1992). In the case of Svp, protein sequence conservation relative to the mammalian cognates (COUP/ARP-1) is much 1403

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higher than for Usp/RXR. The levels of sequence identity between the DNA- and ligand-binding domains are particularly high (>90%; Mlodzik et al., 1990; Ladias and Karathanasis, 1991). This suggests the existence of ligands for Svp and COUP/ARP-1 which are identical or closely related. It is also important to note that the overall conservation between the predicted Svp protein and COUP/ARP-l (77%) is considerably higher than for any other known cognates of developmental regulators in Drosophila and vertebrates. Accordingly, one might expect extensive functional conservation between these proteins, in particular in neural development. However, since the embryonic expression of COUP/ARP-1 has not yet been analysed, no evidence directly supporting this assumption has been obtained. Instead, extensive studies of these transcription factors in vitro and in tissue culture indicate their involvement in the regulation of liver- and intestinespecific gene expression (Sagami et al., 1988; Power et al., 1991; Kliewer et al., 1992b; Tran et al., 1992). Interestingly, the DNA-binding specificities of COUP and ARP-1 are very similar to and overlap with those of RXRs and RARs that are also expressed in the liver (Kliewer et al., 1992b; Mangelsdorf et al., 1992). Moreover, COUP and ARP-1 have been shown to inhibit retinoid receptormediated activities of certain response elements that are activated by RAR/RXR heterodimers and RXR homodimers (Tran et al., 1992). These results suggest an involvement of COUP/ARP-1 in modulating the transcriptional regulation of specific hepatic genes by retinoid receptors (Tran et al., 1992). It is therefore of considerable importance to determine whether such regulatory interactions between COUP/ARP-1 and RARs/RXRs are essential for embryonic development as well. To investigate these issues in vertebrates, the zebrafish (Brachydanio rerio) provides several advantages, e.g. its embryos are translucent and at early developmental stages have a relatively simple central nervous system (Bernhardt et al., 1990; Eisen, 1991). Moreover, retinoids and hormones can be applied to the embryos for detailed investigations of their effects on development or specific target genes. To this end, we have isolated and characterized zebrafish genes related to svp and COUP/ARP-1. The zebrafish cognate of COUP was identified, as well as a novel gene of the svp subgroup of nuclear receptors. Both genes are expressed segmentally in the hindbrain of zebrafish embryos. In addition, their transcripts are regionally distributed within the rostral brain and the developing eyes. These results suggest that the developmental functions of svp-like genes are conserved between vertebrates and Drosophila. The corresponding vertebrate proteins are probably involved in modulating the RAR- and RXRmediated responses to retinoid signals during embryogenesis.

Results Structure of cDNA clones and derived protein sequences Several cDNA clones which cross-hybridize to fragments containing sequences encoding the DNA- and 'ligand'binding domains of the Drosophila svp gene were isolated by low stringency screening of a cDNA library derived from 20-28 h zebrafish embryos (see Materials and methods). Among the positive clones, we identified two independent

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classes of cDNAs, each represented by several independent clones. For each group, a cDNA including the entire protein coding and 3' trailer regions, was identified. Figure 1 shows the complete nucleotide sequence of each of the cDNAs, corresponding to the genes named svp[44] and svp[46], respectively. The svp[44] cDNA is 2937 bp long and contains an open reading frame (ORF) encoding a putative protein of 411 amino acids. Within this ORF, which starts at position 608 and ends with a UGA stop codon at position 1946, the first potential translational initiation site with similarity to the Kozak core consensus (PuCCAUGG; Kozak, 1986, 1987) is located at position 713. Using this initiation site the predicted protein contains DNA- and ligand-binding domains of the Svp type that are particularly closely related to the corresponding domains of the human transcription factors COUP and ARP- 1 (Figure 2). The putative N-terminal sequence is almost identical to the corresponding domains of these two proteins (Figure 2). In addition, Svp[44], COUP and ARP-1 have C-termini at almost identical positions (Figure 2). Sequence determination of the svp[46] clone revealed that this cDNA is 3096 bp long and contains an ORF encoding a protein of 403 amino acids (Figure 1). The predicted N-terminal sequence, which starts at a site with only one mismatch to the Kozak core sequence (Kozak, 1986, 1987), is closely related to those of the transcription factors COUP and ARP-1 (Figure 2). In addition, the relative locations of the regions encoding the presumptive DNA- and ligandbinding domains correspond well to the locations of homologous sequences in the cDNAs of COUP, ARP-l and svp[44] (Figure 2). However, the ORF of svp[46] extends 12-14 amino acid residues beyond the C-termini of the three other proteins (Figures 1 and 2), and the total level of sequence identity between the putative Svp[46] protein and the COUP and ARP-l proteins is -10% lower than in the case of Svp[44] (Figures 2 and 3). Both the svp[44] and svp[46] cDNAs include parts of a poly(A) tail that is preceded by poly(A)+ addition signals (Figure 1), suggesting that complete 3' ends have been identified for both genes.

Sequence conservation between Svp-related proteins Alignments of the predicted zebrafish Svp[44] and Svp[46] protein sequences with Drosophila Svp and the human nuclear receptors COUP, ARP-1 and EAR2, revealed several highly conserved features (Figures 2 and 3). The DNA-binding domains of these six proteins are almost identical (86-100%). The P box region, which is known to be critical for the half-site recognition specificity of members of the steroid receptor superfamily (Umesono et al., 1991), is identical (EGCKS) for Svp and the five related vertebrate proteins. However, the peptide sequence involved in discrimination of half-site spacing (D box) is somewhat variable. While Svp[44], COUP and ARP-1 have identical D boxes (RANRN), Svp[46], EAR2 and Svp had two substitutions each. The large C-terminal ligand-binding domain has a complex structure in which the central part is thought to play an important role in dimerization and regulation of transcription (Forman and Samuels, 1990; Glass et al., 1989). In the case of Svp[44] and Svp[46], high uniform levels of protein sequence identity are present throughout the entire region (89%). This is also observed for all the other members of the Svp/COUP group (>70%; Figures 2 and 3).

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Fig. 1. Nucleotide and predicted protein sequences of the svp[44] and svp[46] cDNAs. (A) For svp[44] the putative protein product is indicated below the DNA sequence in the one letter code, starting with the ATG at nucleotide 713 and ending at 1945. (B) The predicted protein sequence of svp[46] starts with the ATG at nucleotide 650 and ends at 1858. For both cDNA sequences the putative poly(A)' addition signal and the region encoding the DNA-binding domain are underlined.

The variable hinge region separating the DNA- and ligand-binding domains in the steroid receptor superfamily is highly conserved for most of the Svp-related proteins.

Complete identity between the hinge sequences of Svp[44] and COUP provides further support for the assumption that these two proteins are encoded by cognates. Within the hinge

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Fig. 2. (A) Alignment of Svp[44] and Svp[46] with Drosophila Svp (the isoform 'type 1' is shown; Mlodzik et al., 1990) and the human proteins COUP (Wang et al., 1989), ARP-1 (Ladias and Karathanasis, 1991) and EAR2 (Miyajima et al., 1988). The first 108 amino acids of Drosophila Svp and the last five residues of zebrafish Svp[46] are not included. Three or more identical amino acids shared among the six proteins are boxed in black. Similar residues are boxed in grey. The following amino acids were considered to be similar: E,D; V,L,I,M; A,G; F,Y,W; S,T; Q,N. Gaps are indicated by dots. The extent of the DNA-binding domain is highlighted with a bar above the sequence alignments. (B) Alignment of sequences in the 5'UTR of COUP, svp[44] and svp[46]. Vertical lines indicate identical nucleotides. The respective positions within the cDNAs are shown by the numbers flanking the sequences. The COUP 5'UTR sequence is from Wang et al. (unpublished, GenBank accession number M62760).

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Fig. 3. The svp/COUP group of nuclear receptors. (A) Schematic comparison of Svp/COUP-like proteins and related members of the nuclear receptor superfamily. The DNA- and ligand-binding domains are highlighted in black and grey, respectively. Numbers indicate percentages of identical amino acids to the Svp[44] protein within the N-terminal, DNA-binding, hinge region and ligand-binding domains. References are as in Figure 2, except RARca (human RARa; Petkovich et al., 1987) and RXRa (human RXRca; Mangelsdorf et al., 1990). (B) Comparison of whole proteins of the Svp/COUP group. Numbers indicate percentages of identical amino acids over the entire protein length. Note the very high degree of conservation between human COUP and zebrafish Svp[44]. (C) Phylogenetic tree showing relatedness and similarities between the six Svp/COUP group proteins based on an alignment produced by Clustal V (Higgins et al., 1992) and the neighbour-joining method (Saitou and Nei, 1987).

domain, the sequences of Svp[46] and Svp are quite divergent, but the conservation is still as high as 62% and 63%, relative to COUP for these proteins, respectively (Figures 2 and 3). This is in contrast to another member of the Svp group, EAR2, which has only 31% sequence identity to the hinge region of COUP (Figures 2 and 3). While the five related vertebrate proteins have identical N-termini (MAMV), this part of the Svp protein is quite distinct. In addition, the N-terminal regions of the vertebrate proteins are much shorter than Svp, which is > 100 amino acid residues longer (Figures 2 and 3). Where the sequence can be aligned with the vertebrate nuclear receptors, within the second part of the Svp N-terminal domain, the level of sequence identity is 90%) (except for EAR2; 70%) throughout the entire C-terminal region (Figures 2 and 3). Despite these suggestive data, efforts to identify a ligand for COUP have not yet been successful (Power et al., 1991).

Svp-related proteins may regulate neural development in vertebrate embryos

The Drosophila svp gene is required for eye and CNS development (Mlodzik et al., 1990; Y.Hiromi and M.Mlodzik, unpublished results). Based on these observations we have investigated whether similar neural expression patterns have been conserved for any of the vertebrate svprelated genes. For both zebrafish genes, svp[44] and svp[46], analyses of the embryonic expression patterns have confirmed that this is indeed the case. Using in situ hybridization to analyse the spatial distribution of transcripts in whole-mount embryos, both genes were found to be expressed within specific regions of the developing eyes and the neural tube (Figures 4-7).

Svp/COUP-like genes in vertebrate neurogenesis

Expression of the svp[46] gene is first detected in the diencephalic region of the neural keel and the spatial distribution of transcripts, which initially is rather uniform, gradually becomes restricted to the optic stalk region. By contrast, svp[44] seems not to be expressed during the earliest stages when the optic vesicles are formed. Instead, low levels of expression first appear at the posterior edges of the optic vesicles and by the 20 h stage svp[44] transcripts are present within a restricted region of the eye cup. These patterns suggest that both genes may be involved in regionalization of the eye. Other nuclear receptors are also likely to play a role in this process. It has been shown that different levels of retinoic acid (RA) are synthesized along the dorsoventral axis of the retina (McCaffery et al., 1992) and RA treatment of zebrafish embryos induces duplication of the retina (Hyatt et al., 1992). Thus, the RARs expressed in the eye (Dolle et al., 1990) may mediate developmental responses to this ligand. It is not yet clear whether any of the RXRs are also expressed in the developing eye (Mangelsdorf et al., 1992), but the Drosophila RXR homologue usp has been shown to be required for normal eye morphogenesis (Oro et al., 1992). Since COUP and ARP-1 proteins can inhibit retinoid receptor activities on specific response elements (Kliewer et al., 1992b; Tran et al., 1992), these observations indicate the possibility that Svp-related proteins may modulate the effects of retinoids on RXR/RAR-regulated gene expression during eye development in vertebrate embryos. The embryonic expression patterns of both svp-related genes in other regions of the CNS in zebrafish embryos also suggest cross-regulation with RXR/RAR. The early expression within segment-like stripes in the hindbrain region (Figures 4, 5 and 7) is particularly intriguing. These transverse stripes, first detected at 12 h post-fertilization, may correspond to the segmental rhombomere units which become visible at the 15-16 h stage. After the svp[44] and svp[46] transcripts are first detected in the hindbrain, dynamic changes in the expression patterns are observed. While the expression domain of svp[44] extends posteriorly to include most of the hindbrain, svp[46] expression disappears shortly after formation of the rhombomeres. On the basis of the observations that rhombomere formation and the normal pattern of segmental expression of the Krox-20 and Hox-2. 9 genes are sensitive to treatment with RA, the cytoplasmic retinoic acid binding proteins, CRABP I and CRABP II, have been proposed as regulators of hindbrain segmentation (Ruberte et al., 1992). The two murine CRABP genes are regionally expressed in the hindbrain of 8-somite embryos and during the subsequent stages specific rhombomere borders (r3/r4, r4/r5 and r6/r7) are formed at the expression boundaries of these genes (Figure 7; Ruberte et al., 1992). The distribution of transcripts derived from RAR genes correlates also with rhombomere borders (Figure 7). Thus, the segment-like expression of svp[44] and svp[46] in the hindbrain of the corresponding developmental stages of zebrafish and the biochemical data from previous studies suggest that these genes are also involved in modulating the retinoid signals necessary for the proper generation and/or specification of rhombomeres. While CRABP I and II are probably involved in regulating the levels of free RA available for binding to the nuclear receptors, the Svp-related proteins may play a more specific role by competing with RARs

and RXRs for binding to RAREs of different subsets of retinoid responsive genes. The restricted svp[44] expression in the rostral brain (Figure 4) is reminiscent of the regional expression reported for the pax[zf-a] gene (Figure 7; Krauss et al., 1991a,b). By 20 h post-fertilization, both genes have overlapping expression on the diencephalon. Interestingly, murine RXRy is also expressed within a restricted region of the forebrain during embryogenesis (Mangelsdorf et al., 1992). These observations suggest that regulatory interactions between Pax, RXR/RAR and svp-like genes might be important for regionalization of the rostral brain. A similar relationship may exist in the eye (Figures 6 and 7; see above) where members of these gene families also have overlapping expression patterns. Svp-related genes of vertebrates may play a role in mesoderm differentiation Both svp[44] and svp[46] are expressed in mesodermal tissues of zebrafish embryos, but the spatial distribution of

their transcripts is completely different. While svp[46] transcripts are detected in somites (Figure 5), svp[44] is expressed in the intermediate mesoderm (Figure 4A) which ultimately gives rise to the kidneys and other parts of the urogenital system. In both cases, mesodermal expression is most intense in early embryonic stages, indicating transient roles in the specification of different tissues. Various Pax, RAR, RXR and CRABP genes are also differentially expressed in somitic and intermediate mesoderm during embryogenesis (Deutsch et al., 1988; Dolle et al., 1990; Dressler et al., 1990; Krauss et al., 1991c; Mangelsdorf et al., 1992), suggesting similar cross-regulatory interactions as proposed for the developing brain.

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Conclusions The results presented in this paper provide the first evidence that members of the Svp/COUP group of nuclear receptors are expressed during vertebrate development. The expression patterns suggest a functional conservation to their Drosophila homologue in neurogenesis and eye development. Moreover, the observed correlation with the segment-related hindbrain expression patterns of RARs/ CRABPs and the known potential of Svp-like proteins in modulating the transcriptional activation mediated by RARs/RXRs imply that they are involved in the complex gene network responsible for the interpretation of retinoid signals during vertebrate development.

Materials and methods Embryos Zebrafish were maintained and bred essentially as described by Stuart et al. (1988). Developmental age is given in hours after fertilization at 28.5°C, which was the temperature of incubation.

Cloning

Approximately 1.5 x 106 plaques of a 20-28 h zebrafish embryonic XZAP-ll cDNA library were screened by plaque hybridization at low stringency (McGinnis et al., 1984) using a mix of probes derived from the regions of the svp gene (Mlodzik et al., 1990) encoding the DNA- and ligandbinding domains. Several independent clones derived from the svpL44] and svp[46] genes were isolated. The cDNA inserts were subcloned by helper phage cotransfection (Stratagene).

Sequencing and sequence analysis DNA sequence was determined by the chain termination method on double-stranded templates (Sanger et al., 1977) using Sequenase (United

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States Biochemical Corp.). Overlapping deletion clones of cDNAs svp[44] and svp[46] were sequenced on both strands (except for nucleotides 2383-2645 of the 3' untranslated region of svp[44]). Sequence assembly and analysis were done with programs from the GCG software package 7.1 (Devereux et al., 1984). FASTA (Altschul et al., 1990) was used to perform database searches. In situ hybridization analysis In situ hybridization to whole-mount embryos was performed according to the protocol described by Krauss et al. (1991b). Stained embryos were analysed with DIC optics in a Nikon Microphot-FXA microscope.

Acknowledgements We thank Robert Riggleman, Kathryn Helde and David Grunwald for generously providing the embryonic cDNA library. We are also grateful to Desmond Higgins and Peter Rice for help with the computer analysis and Anne Ephrussi for helpful comments on the manuscript. Financial support was obtained from the Norwegian Research Council for Science and the Humanities, Norwegian Cancer Society and Nansen Foundation.

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Received on December 16, 1992

Note added in proof The sequence data of sup[44] and sup[461 have been deposited in the EMBL Data Library under the accession numbers X70299 and X70300, respectively.