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The Mammalian Odz Gene Family

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The Mammalian Odz Gene Family Developmental Biology 217, 107–120 (2000) doi:10.1006/dbio.1999.9532, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE The Mammalian Odz Gene Family: Homologs provided by Elsevier - Publisher Connector of a Drosophila Pair-Rule Gene with Expression Implying Distinct yet Overlapping Developmental Roles Tali Ben-Zur, Erez Feige, Benny Motro, and Ron Wides1 Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900 Israel The Drosophila pair-rule gene odz (Tenm) has many patterning roles throughout development. We have identified four mammalian homologs of this gene, including one previously described as a mouse ER stress response gene, Doc4 (Wang et al., 1998). The Odz genes encode large polypeptides displaying the hallmarks of Drosophila Odz: a putative signal peptide; eight EGF-like repeats; and a putative transmembrane domain followed by a 1800-amino-acid stretch without homology to any proteins outside of this family. The mouse genes Odz3 and Doc4/Odz4 exhibit partially overlapping, but clearly distinct, embryonic expression patterns. The major embryonic sites of expression are in the nervous system, including the tectum, optic recess, optic stalk, and developing eye. Additional sites of expression include trachea and mesodermally derived tissues, such as mesentery, and forming limb and bone. Expression of the Odz2 gene is restricted to the nervous system. The expression patterns suggest that each of the genes has its own distinct developmental role. Comparisons of Drosophila and vertebrate Odz expression patterns suggest evolutionarily conserved functions. © 2000 Academic Press Key Words: odz (odd Oz); pattern formation; central nervous system (CNS); embryonic development; eye. INTRODUCTION of the Hedgehog (Hh) pathway (see Johnson and Scott, 1998) and as a constituent of the shared receptor tyrosine kinase odd Oz (odz, or Tenm) is a novel gene involved in (RTK) signaling pathway (see Rubin, 1989; Diaz-Benjumea directing segmentation in Drosophila melanogaster (Levine and Hafen, 1994). These expression data have been corrobo- et al., 1994, 1997a,b; Baumgartner et al., 1994). odz has been rated by genetic interactions and molecular inductions designated a pair-rule gene, in that embryos mutant for the between odz and known Hedgehog and RTK pathway gene lack every second (odd-numbered) body segment. odz constituents (Levine et al., 1997b; Weiss and Wides, unpub- is unique in that it is the only pair-rule gene discovered lished results). which encodes a protein involved in signal transduction on odz is a more than 120-kb gene, with many sophisticated the cell surface, rather than in transcription. This gene also levels of expression control. The Odz protein predicted by directs the development and differentiation of the embry- the mature 11-kb message’s open reading frame is a mole- onic central nervous system (CNS), dorsal vessel (heart), cule of 2731 amino acids in length (Levine et al., 1997a). trachea, and hemocytes. The gene displays further pluripo- The conceptually translated protein bears a putative signal tency in Drosophila pupae, where it is expressed in the peptide followed by eight EGF-like (epidermal growth fac- imaginal precursors of many adult structures. Especially tor) repeats (see Fig. 1). The post-EGF region of Odz striking is the expression of odz in the morphogenetic (C-terminally from the EGF repeats) contains a hydrophobic furrow and in photoreceptor R7 cells in eye imaginal discs sequence which predicts a potential transmembrane do- and adjacent to compartment boundaries in wing imaginal main (Kyte and Doolittle, 1982) and is followed by a discs. The nature of odz expression in these imaginal number of tyrosine kinase (TK) phosphorylation consensus tissues (Levine et al., 1997b) implicates odz as a constituent sites (Levine et al., 1994) in a nearly 1800-amino-acid stretch with no SH2 or SH3 motifs (Koch et al., 1991). The 1 To whom correspondence should be addressed. Fax: 972 3 535 region of the Odz protein C-terminal to the EGF repeats is 1824. E-mail: [email protected]. in fact tyrosine phosphorylated (Levine et al., 1994; Dgany 0012-1606/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 107 108 Ben-Zur et al. FIG. 1. Schematic of Odz gene homologies. The C. elegans, D. melanogaster, three mouse (Odz2, Odz3, and DOC4/Odz4), and one human (ODZ1) Odz homologs are aligned schematically. In addition, the sequenced portion of the human ODZ3 gene is depicted, in order to complement the incomplete mouse Odz3 (over the more than 1000 overlapping amino acids, ODZ3 and Odz3 proteins are 98% identical). Putative signal peptides and transmembrane domains are marked as shaded boxes, EGF-like repeats as shaded ovals, and tyrosine phosphorylation consensus sites by the symbols PO4-Y. Thin dashed lines with arrows represent uncloned portions of the genes, based on expectations from Northern blots and analogy to the full-length genes. The scale bar represents 500 amino acids; each expected polypeptide is approximately 2800 aa. Accession numbers are AF195418 (Odz3), AF195419 (Odz2), and AF195420 (Odz3). and Wides, unpublished), which indicates that this region is Broad evolutionary conservation has been observed for intracellular. We envision Odz as a transmembrane protein Drosophila genes responsible for segmentation and seg- which contains EGF repeats capable of mediating an extra- ment identity (homeotic genes). However, while the ho- cellular protein–protein interaction and which is intracel- meotic gene framework is functionally conserved among all lularly processed and phosphorylated on tyrosine. There are animals, the framework for genes which direct segmenta- no proteins with homology to the entire Odz sequence. tion has not been elucidated in vertebrates. Nonetheless, However, a tenuous structural analogy can be drawn be- vertebrate homologs of essentially all Drosophila segmen- tween Odz and the protein Notch (see Artavanis-Tsakonas tation genes exist, and virtually all play roles in early et al., 1995). Notch is an EGF repeat containing transmem- development. Some Drosophila segmentation gene ho- brane protein whose intracellular domain is cleaved, for mologs are involved in the earliest stages of patterning in proposed subsequent translocation to the nucleus, in re- vertebrate embryos (see Beddington and Robertson, 1999), sponse to extracellular signals (see Lieber et al., 1993; and while others have their Drosophila postsegmentation roles Struhl and Adachi, 1998). Some aspects of Odz posttransla- conserved (see Hogan, 1999). For yet others, such as hairy, tional modifications and signaling may prove to mirror there is first evidence that the genes will prove to play some Notch signaling (Dgany and Wides, unpublished). role in establishing segment number (Muller et al., 1996). Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. Mammalian Odz Gene Family 109 Through EST database searches, medium stringency hy- clones (dJ799F15 and dA83A7) are likely to contain exons compris- bridization screens, and PCR methods, we have identified ing only up to 30 codons. ODZ1 stretches over approximately 700 and cloned three Odz homologs in mouse. A fourth ho- kb of genomic DNA and is presumably transcribed as a greater than molog identified is a human sequence extracted from Hu- 10-kb message. man Genome Project genomic sequences, as recognized by sequence similarity. One of cloned homologs from mouse Computer Analysis of Sequence has been reported recently under the name Downstream of CHOP 4 (DOC4) (Wang et al., 1998). We have isolated these Analysis of sequences was carried out using a Wisconsin Genet- genes and have characterized their expression patterns. The ics Computing Group software package (versions 9 and 10) on a dedicated Unix workstation. This included sequence assembly, expression patterns suggest that each of the genes has database comparisons (FASTA of Pearson and Lipman, 1988; pleiotropic developmental roles which are distinct from BLAST of Altschul et al., 1990), conceptual restrictions, conceptual those of the other family members. The expression patterns translations, protein motif searches, and hydropathy analysis. also imply that the developmental roles of Odz are evolu- Blasting was also carried out (at www.ncbi.nlm.nih.gov/cgi-bin/ tionarily conserved from flies to mammals. BLAST/), and PSORT II was utilized (at psort.nibb.ac.jp). MATERIALS AND METHODS Northern Analyses Tissues were extracted (as in Motro et al., 1991). The RNA was Cloning Odz cDNAs subjected to electophoresis in formaldehyde containing gels and was transferred to Magna nylon membranes (MSI). The filters were EST database searches using the 39 portion of the Drosophila odz hybridized to 32P-labeled random-primed cDNA gene probes under sequence (and C-terminal portion of the Odz protein for translated standard conditions (Sambrook et al., 1989) and then exposed to BLAST searches) revealed several significant matches. The EST X-ray film. The sizes of the genes’ transcripts were determined in clone 789682 was obtained and was used for hybridizations at comparison to known markers. medium stringency, and also at high stringency, on cDNA libraries from 11.5-dpc (days postcoitum) mouse embryos (as in Levine et al., 1994). This initial screen yielded partial cDNA clones of Odz2, In Situ RNA Analysis Odz3, and Odz4 (Doc4). The resulting clones were then sequenced and utilized to progress in the 59 direction of the cDNAs by In situ RNA hybridization was carried out on embryonic and carrying out reiterative, high-stringency screens for each of the adult 10-mm cryosections as previously described (Motro et al., three genes. Initially, the library used was a fetal poly(T)-primed 1991). As an antisense probe, an EcoRI subclone of Odz3 (clone 1 library (kind gift of Maria Rozakis-Adcock), and then a random- 917V) in KS (Stratagene), spanning nucleotides 3400 to 2300 bp primed/poly(T)-primed mixed 15-dpc embryo library (Clonetech, from the 39 end of the Odz3 message, was linearized at the PstI 35 Cat.
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