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Genomics 88 (2006) 185–195 www.elsevier.com/locate/ygeno

Male-biased expression of X-chromosomal DM domain-less Dmrt8 in the mouse

Anne-Marie Veith a,1, Jürgen Klattig b,1, Agnes Dettai a, Cornelia Schmidt a, ⁎ Christoph Englert b, Jean-Nicolas Volff a,

a Department of Physiological Chemistry I, Biozentrum, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany b Leibniz Institute for Age Research–Fritz Lipmann Institute, Jena, Germany

Received 11 December 2005; accepted 14 January 2006 Available online 20 February 2006

Abstract

The vertebrate DMRT family encodes putative factors related to the sexual regulators () and MAB-3 (). They share a highly conserved DNA binding motif, the DM domain. In human and mouse seven DMRT genes (DMRT1–DMRT7) have been analyzed. DMRT8, a gene related to DMRT7, is located on the X in placental mammals. While DMRT8 is single copy in most mammals, three copies are present in mouse, rat, and rabbit. Despite the loss of the DM domain, DMRT8 genes have been maintained in the mammalian lineage, suggesting a DM domain-independent function. In adult mouse, two Dmrt8 genes are expressed exclusively in testis. Dmrt8.1 mRNA was detected in Sertoli cells by in situ hybridization. In embryos, Dmrt8.2 shows a dynamic expression restricted to male and female gonads and might therefore be involved in sexual development in the mouse. © 2006 Elsevier Inc. All rights reserved.

Keywords: DM domain; DMRT; DMRTC1; Sex determination; Sex differentiation; Sertoli cells; Testis; X chromosome

Introduction modules, the DM domain binds into the minor groove of the DNA [3,4]. Over the past few years the DMRT gene family (DMRT: The most prominent member of the vertebrate DMRT gene DSX- and MAB-3-related ) has come to the family is DMRT1. In human, DMRT1, DMRT2, and DMRT3 are fore because some of its members are involved in sex physically clustered on chromosome 9p24. Mutations and determination/differentiation in several metazoan phyla, repre- deletions in this region are associated with male-to-female sex senting the first known examples of conserved sexual reversal [5–7]. DMRT1 has been suggested to play a role during regulatory genes [1,2]. Members of the DMRT gene family sexual development in a variety of vertebrate species, including share a highly conserved motif, the DM domain. The fish [8–12], frogs [13], lizards and turtles [14,15], birds [16– DM domain is a small DNA binding motif that contains two 18], and mammals [5,19–21]. In the teleost fish medaka distinct parts: first, an N-terminal DNA binding part contain- (Oryzias latipes), two genes are present. In addition to ing six cysteines and two histidines at invariant positions, dmrt1a, which is clustered together with dmrt2 and dmrt3 on forming two intertwined zinc-finger-like DNA binding linkage group 9 [10], an additional copy named dmrt1by or dmy modules, and second, a C-terminal disordered tail, which is located in the sex-determining region on the Y chromosome, acts as recognition helix for DNA binding. Unlike classical but not on the X chromosome. This copy shows attributes of a recently evolved master male sex-determining gene [22–25]. No orthologue of dmrt1by was found in more distantly related ⁎ Corresponding author. Fax: +49 931 888 4150. fish species, indicating that this gene is not a general master sex- E-mail address: [email protected] (J.-N. Volff). determining gene in fish [26,27]. In birds, which have a 1 These authors contributed equally to this work. chromosomal sex-determining system with ZZ males and ZW

0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2006.01.003 186 A.-M. Veith et al. / Genomics 88 (2006) 185–195 females, DMRT1 is located on the Z chromosome but not on the Partial DMRT7- and DMRT8-encoding sequences were W. The expression of DMRT1 in the genital ridges prior to detected for several other placental mammals (Table 1). In gonadal differentiation, with a higher expression in male marsupials (opossum Monodelphis domestica) and monotremes embryos, is compatible with a role for DMRT1 as the male ( Ornithorhynchus anatinus), only Dmrt7 could be sex-determining gene in birds [16–18]. The observation that identified. In nonmammalian species neither Dmrt7 nor Dmrt8 DMRT1 is Z-linked in birds and that the Y-chromosomal could be found despite using all available sequence resources, dmrt1by is the male-inducing factor in the fish medaka suggests suggesting that these genes are mammalian-specific. that in some nonmammalian species a DMRT1 gene has played Most database information available for DMRT7- and arolecomparabletothatofthemammalianmalesex DMRT8-encoding sequences corresponded to four distinct determination gene SRY (sex-determining region on the Y) regions, which are conserved in DMRT7 and DMRT8 among during the evolution of sex [18,24,25]. species (regions I–IV, Table 1, Fig. 1a). Region I (75 aa) and Recent analysis of other members of the DMRT gene region II (52 aa) include the DM domain (Figs. 1b and 1c). family in mouse (Mus musculus), chicken (Gallus gallus), frog Regions III (35 aa) and IV (48 aa) are located downstream of the (Xenopus laevis), and fish (medaka, zebrafish Danio rerio) putative DM domain in the carboxy-terminus of DMRT7 and indicated that they show restricted and nonoverlapping DMRT8. Region III and region IV are conserved (48–60% expression patterns during embryogenesis. DMRT2 in mouse, amino acid similarity) between DMRT7 and DMRT8, but not in chicken, and fish has an expression pattern consistent with a other DMRT , thus confirming a close relationship function during somitogenesis [29–32]. DMRT3 is expressed in between DMRT7 and DMRT8 [2–35]. a subset of dorsal interneurons in mouse, chicken, and medaka [31,33] and DMRT4 is expressed in the developing olfactory DMRT8 is located on the X chromosome in mammals and system in medaka as well as in the frog X. laevis [31,34]. triplicated in rabbit and rodents Expression of Dmrt5 is detectable during embryogenesis in the brain of mouse [30]. In addition to dmrt1, some other DMRT In all species for which information about chromosomal genes also show gonadal expression in fish. Medaka dmrt2–4 location is available (human, chimpanzee, mouse, rat, and dog), share an expression in larval and adult gonads, but not during the DMRT8 genes are located on the X chromosome, but not early gonadal development [31]. on the Y chromosome, while DMRT7 is always located on In mouse, three DMRT genes other than Dmrt1 (Dmrt3, autosomes (Table 1). This suggests that the common ancestor of Dmrt4, and Dmrt7) show an expression pattern consistent with all mammalian DMRT8 genes was X chromosomal. In contrast a role in gonadal development [30]. Dmrt3 is expressed in to the situation observed for most mammals, three Dmrt8 genes interstitial cells of the testis. Dmrt4 is detected in gonads of both are present in rabbit, mouse, and rat. Phylogenetic analysis sexes and Dmrt7 is expressed in somatic and germ cells in the suggests that serial events of duplication occurred in the rabbit ovary, but solely in germ cells in testis [30]. Partial redundancy and rodent lineages after their divergence from other mammals, of these genes with Dmrt1 during gonadal development might approximately 77 million years ago [36]. In rodents, triplication explain the relative mild phenotype observed in Dmrt1-null of Dmrt8 arose before the split between rat and mouse (Fig. 1d). mutant mice [20,30]. The three Dmrt8 genes in mouse form an X-linked Dmrt8 gene Another putative DMRT gene related to DMRT7 called cluster (Fig. 2a). DMRT8 (aka DMRTC1) is present on the X chromosome in human and mouse [35]. In contrast to all other known DMRT Structural comparison and coding potential of DMRT7 and genes, the DM domain-encoding sequence of DMRT8 is not DMRT8 conserved in human and mouse. Based on comparison of human cDNA and genomic DMRT8 sequences, it has been Sequence comparison of DMRT7 genomic and cDNA doubted whether DMRT8 has the potential to encode functional sequences showed that the DM domain-encoding sequence is DM domain-containing proteins in any species [30,35]. separated by a small intron (Figs. 1a and 2b, size of this intron is Here, we report the presence of several Dmrt8 genes on the 83 bp in human and 74 bp in mouse). Analysis of region I and of X chromosome of mouse and analyze their expression and region II (Figs. 1b and 1c) revealed that in DMRT8 a putative evolution in the mammalian lineage. DM domain-encoding sequence is present and conserved between most investigated species (50–60% amino acid Results similarity). Similar to DMRT7, an intron separates the DM domain-encoding sequences of DMRT8 (81 bp in mouse DMRT7 and DMRT8: two putative mammalian-specific DMRT Dmrt8.2). Thus, in DMRT7 and DMRT8, two separate exons genes encode the putative DNA-binding modules and disordered tail of the DM domain (Figs. 1b and 1c). The presence of an intron DMRT7 and DMRT8 sequences from human and mouse in the DM domain-encoding sequence is not unique for DMRT7 [30,35] were used as initial queries in a BLAST analysis of and DMRT8, since a similar situation has been also described public sequence databases (Table 1). Complete sequences for for DMRT1 and medaka dmrt1bY [10,12,22]. However, the both DMRT7 and DMRT8 were identified in the genome position of this intron is not conserved in DMRT1 and DMRT7/ drafts of chimpanzee, rhesus monkey, dog, cow, mouse, and rat. 8, suggesting an independent origin. A.-M. Veith et al. / Genomics 88 (2006) 185–195 187

Table 1 Dmrt7 and Dmrt8 genes in mammals Gene Organism Common name Region Chr. Accession No. I II III IV DMRT7 Placental mammals Homo sapiens Human + + + + 19 NM_033052a Pan troglodytes Chimpanzee + + + + 19 XM_512691a Pongo pygmaeus Orangutan (+) + + + 830215935 (I, II), 610853793 (III), 769304430 (IV)b Macaca mulatta Rhesus monkey + + + + 125119: 54063-61607c Mus musculus Mouse + + + + 7A3 NM_0227732a Rattus norvegicus Rat + + + + 1q21 XM_218456a Cavia porcellus Guinea pig + + + 815550916 (I, II)b Oryctolagus cuniculus Rabbit + + 610853793 (III), 641763333 (IV)b Canis familiaris Dog + + + + 1 1: 114631055-1: 114635019c Felis catus Cat + + 717147123 (III), 643810631 (IV)b Bos taurus Cow + + + + 111850: 35-5132b Sus scrofa Pig + 470951814 (II) Loxodonta africanus Elephant + 520362633 (III)b Echinops telfairi Tenrec + + 691874606 (III), 664728419 (IV)b Dasypus novemcintus Nine banded armadillo + + 548706168 (II), 548709736 (IV)b Marsupials Monodelphis domestica Opossum + + + + 12174: 15725-20784c Monotremes Ornithorynchus anatinus Platypus + + + 739243735 (I), 639540765 (II), 814398338 (IV)b DMRT8 Placental mammals H. sapiens Human ~ + + + Xq13 NM_033053, AJ291670a P. troglodytes Chimpanzee ~ + + + X X: 74147993-74151740c Po. pygmaeus Orangutan + + + 745337619 (I, II), 757226430 (III), 834010957 (IV)b Ma. mulatta Rhesus monkey ~ + + + 35645: 10144-15652 b C. familiaris Dog + + + + X XM_549077a F. catus Cat + + + + 662213964 (I, II), 644382180 (III), 653045346 (IV)b B. taurus Cow + + + + 292083: 1499-4257c S. scrofa Pig + + + 812759849 (I, II), 812459919 (III)b Echinops telfairi Tenrec + 776320897 (III)b D. novemcintus Nine banded armadillo + + 558920343 (III), 596223373 (IV)b Dmrt8.1 M. musculus Mouse + + + XD XM_027591a R. norvegicus Rat + + X NM_001025288a Dmrt8.2 M. musculus Mouse ~ + + + XD XM_205213a R. norvegicus Rat + + + Xq13 XM_228580a Dmrt8.3 M. musculus Mouse ~ + + XD XM_356345a R. norvegicus Rat ~ + + Xq13 NM_001014222a Dmrt8.1 O. cuniculus Rabbit + + + AAGW01043754 (WGS)a Dmrt8.2 O. cuniculus Rabbit ~ + 616672842 (I, II)b Dmrt8.3 O. cuniculus Rabbit + + 638708812 (I, II)b DMRT7 and DMRT8 sequence sources: a http://www.ncbi.nlm.nih.gov/, bWGS (whole genome shotgun) or EST (expressed sequence tag) trace sequences (http:// www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml), cnucleotide sequence positions of DMRT7 and DMRT8 genomic sequences in the genome drafts available at the UCSC genome browser (http://genome.uscs.edu/). Sequences encoding regions I, II, III, and IV are conserved in DMRT7 and DMRT8 (Fig. 1a). Regions I and II constitute the DM domain. Sequences encoding a truncated version of the DM domain are indicated with ∼; partial, incomplete WGS sequences for region I are in parentheses. Chr., chromosomal location.

Based on their common sequence characteristics, we iden- vestigated primate species indicates that the donor splice site tified four orthologous groups of DMRT8 sequences (Fig. 1d). downstream of the zinc-finger module-encoding exon (region I) The first group consists of DMRT8 from primates (apes and is mutated (GT to AT). A putative translation start site has been other Old World monkeys). All amino acids at invariant identified downstream of the DM domain-encoding region [35] positions of the DM domain are present, but in genomic DNA (Fig. 1c). Therefore, the primate DMRT8 genes do not have the from the three apes (human, chimpanzee, and orangutan) the potential to encode a DM domain. open reading frame 5′ of the potential DM domain of DMRT8 is In contrast to the situation observed in primates, this splice disrupted by a stop codon (Fig. 1b). In the human DMRT8 site is intact in cat, dog, cow, and pig. In these species, one of cDNA sequence AJ291670 [35], the intronic sequence that the invariant histidines within the DM domain has changed separates the DM domain is retained in the cDNA. Conse- either to glutamine or to glutamic acid. However, a second quently, the open reading frame is disrupted by a stop codon histidine is present next to this position (Fig. 1b). So far, within the intronic sequence. Sequence comparison of human this histidine is found only in Dmrt7 of opossum and in DMRT8 cDNA and genomic DNA of DMRT8 from all in- the investigated Dmrt8 sequences. Whether this histidine can 188 A.-M. Veith et al. / Genomics 88 (2006) 185–195 A.-M. Veith et al. / Genomics 88 (2006) 185–195 189 compensate for the loss of the first one is unclear. Thus, the decreased from E14.5 to E15.5 and reappeared exclusively in unique Dmrt8 gene in cat, dog, cow, and pig might encode a testis after birth. protein with a DM-like domain, but whether it binds to DNA The spatial expression pattern of Dmrt8 genes in adult mice remains to be investigated. was analyzed by RNA in situ hybridization on testis sections. The third group of sequences consists of the three Dmrt8- Dmrt8.1 mRNAwas detected only in Sertoli cells, a somatic key encoding sequences from rabbit. As observed in some other cell type in testis (Fig. 4b). The -specific expression placental mammals, one of the invariant histidines of the DM was confirmed by comparison to the expression pattern of Wt1 domain is substituted (Fig. 1b). Finally, three different Dmrt8- (Fig. 4c), a marker for Sertoli cells in adult mice [37]. We could encoding sequences are present in rodents. Dmrt8.2 (mouse) not detect Dmrt8.2 expression by in situ hybridization in adult and Dmrt8.3 (mouse and rat) encode truncated versions of the testis, although expression was clearly detectable by RT-PCR. zinc-finger module-containing part of the DM domain (Fig. 1b), This might be due to a lower expression of Dmrt8.2 compared while this sequence seems to be completely absent from mouse to Dmrt8.1. and rat Dmrt8.1. In mouse and rat, a LINE element is inserted The three Dmrt8.1 and Dmrt8.2 isoforms derived from into the putative zinc-finger module-encoding exon of Dmrt8.1, different PCR products of testis (Fig. 2c) were cloned and giving a hint about the degeneration of this module (Fig. 2b). analyzed. For Dmrt8.1 and Dmrt8.2, eight coding exons, and The second part of the DM domain (region II) is present in all nine exons for Dmrt7, are present (Fig. 2b). Comparison of three rodent Dmrt8 sequences (Fig. 1c). Dmrt8.1 genomic sequences from rat and mouse to other mammalian Dmrt8 sequences indicates that the 3′ alternative Male-biased expression and of Dmrt8.1 terminal exon 7 of Dmrt8.1 is apparently specific in mouse. In and Dmrt8.2 in the mouse addition, exon 6 of Dmrt7 is absent in all three Dmrt8 genomic sequences (Fig. 2b, Dmrt8.3 not shown). None of the Dmrt8.1 Expression of Dmrt8 genes in adult mice was analyzed by isoforms encodes the first part of the DM domain because this RT-PCR using several organs (Fig. 2c). In contrast to human exon is broken, most likely due to the insertion of a LINE DMRT8, which is expressed in various organs, including brain, element (Fig. 2b). Exon 2 (DM domain part II) is also absent in lung, kidney, pancreas, and gonads of both sexes [35], Dmrt8.1b and Dmrt8.1c (Fig. 2c). Here, exon 3 is elongated 5′ expression of Dmrt8.1 and Dmrt8.2 was exclusively detected of the normal splice site (exon 3′) and contains a novel predicted in testis of adult mice. For both Dmrt8 genes, three different translation start site. Dmrt8.2c encodes a truncated version of isoforms could be identified (Fig. 2c). All these isoforms are the first part of the DM domain, while the intron that separates also expressed in mouse embryonic testis. Expression of the two DM domain-encoding exons is present in Dmrt8.2b, Dmrt8.3 could not be detected by RT-PCR in adult mice and and similar to the situation in humans, a stop codon adjacent to embryos using several primer pairs (not shown). Whether the 5′ splice site disrupts the open reading frame (Fig. 2c). In Dmrt8.3 is a pseudogene has to be further investigated. summary, none of the investigated Dmrt8 isoforms encodes the Embryonic expression of Dmrt8.1 and Dmrt8.2 was assessed complete zinc-finger modules of the DM domain, suggesting by quantitative real-time PCR (qRT-PCR) of cDNA from that they probably do not have the DNA-binding capacity different tissues of single embryos at embryonic day (E) 13.5 conferred by this domain. with primers recognizing all Dmrt8.1 and Dmrt8.2 isoforms. While Dmrt8.1 is exclusively expressed in testis of adult mice Discussion (Fig. 2c), expression could be detected at low levels in all embryonic organs tested with no apparent sex-specific expres- Evolution of DMRT7 and DMRT8 in the mammalian lineage sion pattern (Fig. 3a). In contrast to Dmrt8.1, expression of Dmrt8.2 in embryos was detectable exclusively in testis and Based on currently available data from public databases, ovary, with a substantially higher expression in testis at E13.5 DMRT7 and DMRT8 are present in mammals, but not in (Fig. 3b). To investigate the temporal expression of Dmrt8.2 nonmammalian vertebrate species, including chicken, frogs, during gonadal development, qRT-PCR was performed on and teleost fishes. Thus, these two DMRT genes might be cDNA from male and female urogenital ridges/gonads starting mammalian-specific. The structural and sequence similarity of from E8.5 to 5 weeks after birth (Fig. 3c). Dmrt8.2 shows a DMRT7 and DMRT8 is consistent with the hypothesis that dynamic expression for only a short period during gonad DMRT8 originated by duplication of DMRT7 [35].The development and is first visible from E11.5 to E12.5. At E13.5, presence of DMRT7 in monotremes, marsupials, and placental Dmrt8.2 expression showed a peak in testis, while expression in mammals suggests that DMRT7 was probably formed after the ovary strongly decreased. Expression of Dmrt8.2 in testis also divergence of mammals from other vertebrates and before the

Fig. 1. Organization and conserved domains in mammalian DMRT7 and DMRT8 proteins. (a) Schematic organization of DMRT7 and DMRT8 proteins. Regions I, II, III, and IV are conserved in DMRT7 and DMRT8. Region I and region II constitute the DM domain. The DM domain-encoding sequence is separated by an intron (arrowhead). (b, c) Sequence comparison of DMRT7 and DMRT8 putative DM domain translational products. The boundary between region I (DNA binding) and region II (disordered tail), which are encoded by different exons, is labeled with an arrowhead. In DMRT8 from human, chimp, and orangutan, the open reading frame is disrupted by a stop codon 5′ of the DM domain (indicated by a star). Only a partial DMRT7 sequence of region I is currently available for orangutan. (d) Phylogenetic analysis of DMRT7 and DMRT8. The tree, based on combined alignments from regions II, III, and IV (116 aa, bootstrap-neighbor-joining, 1000 pseudosamples), is unrooted. Sequences present only for regions III and IV were not included. Accession numbers are given in Table 1. 190 A.-M. Veith et al. / Genomics 88 (2006) 185–195

Fig. 2. Genomic organization and testis-specific expression of Dmrt8 genes in adult mouse. (a) X-chromosomal localization of Dmrt8.1, Dmrt8.2, and Dmrt8.3 in the mouse. (b) Genomic organization of Dmrt7, Dmrt8.1, and Dmrt8.2. Primers used are indicated as arrows. Dmrt8.2 encodes a truncated version of the DM domain. (c) RT-PCR-analysis of Dmrt8.1 and Dmrt8.2 isoforms in organs of adult mice and embryonic testis from E13.5. Tbp (TATA box-binding protein) was used as a control. As a negative control, water instead of cDNA was added to the PCR. The following primers were used: Dmrt8.1a, f1, r1; Dmrt8.1b, c f3, r3; Dmrt8.2b, f1, r3; Dmrt8.2b, c, f4, r3 (r4 was used for sequencing the 3′ end of Dmrt8.2a, b, c). Expression of Dmrt8.3 was not detectable (not shown). The putative translation start sites are indicated as arrows. emergence of placental mammals approximately 210–310 duplication event that led to the formation of DMRT8 might million years ago [38]. Based on the current accessible data, have occurred either before the appearance of placental the widespread distribution of DMRT8 in placental mammals mammals or even within the placental mammalian lineage. suggests that this gene is at least 100 million years old. The Additional data, mainly from other marsupials and from A.-M. Veith et al. / Genomics 88 (2006) 185–195 191

Fig. 3. Quantitative real time RT-PCR analysis of Dmrt8.1 and Dmrt8.2 expression in mouse embryos. (a, b) Determination of Dmrt8.1 and Dmrt8.2 expression levels in different tissues at E13.5. (c) Determination of Dmrt8.2 expression levels at different stages of gonad development. p1, 1 day after birth; w3, 3 weeks after birth; w5, 5 weeks after birth. All experiments were performed at least twice. In each case, the result of one representative experiment is shown. To define a point of reference for the y axis, the expression level in adult testis was set as 1. Probably due to slight age differences between the embryos used in panels b and c, there is a difference between male and female expression levels of Dmrt8.2 at E13.5 in panels b and c. platypus, will be necessary to determine the evolutionary origin during evolution. Most investigated species of DMRT8. have one copy of DMRT8, while at least three copies are present Our data suggest that the common ancestor of all placental in rabbit, mouse, and rat. Thus, Dmrt8 was duplicated several mammalian DMRT8 genes was located on the X chromosome. times in the rabbit, mouse, and rat lineage probably after their No homologue of DMRT8 was found on the Y chromosome, divergence from other mammals. The Dmrt8 copies might have indicating that DMRT8 has been subsequently lost from the Y been generated through two possible scenarios. Three Dmrt8 chromosome or was formed specifically on the X chromosome copies were already present in the common ancestor of mouse, 192 A.-M. Veith et al. / Genomics 88 (2006) 185–195

Fig. 4. RNA in situ hybridization on paraffin sections of mouse adult testis. Hybridization with (a, b) Dmrt8.1 and (d) Wt1 antisense probes shows expression of both genes in Sertoli cells (marked by arrows, a higher magnification is shown in panel b). Wt1 is a marker for expression in Sertoli cells, a somatic cell type located in the outer layer of testis cords that surrounds the spermatogonia [37]. (c) No signal was detected using the Dmrt8.1 sense probe as a control. rat, and rabbit. Subsequently, these copies might have been regions correspond to functional domains involved in dimer- homogenized through gene conversion independently in the ization and recruitment of coregulatory factors. Dimerization rabbit and the mouse/rat lineages. Alternatively, the three Dmrt8 seems to be important for DNA binding of the metazoan DM genes were formed by independent duplications after the domain [3,40]. Therefore, DM domain-like or DM domain-less separation of rabbit and rodents, approximately 55 million years DMRT8 proteins could evolve as dominant-negative regulators ago [36]. as they might be able to form heterodimers with DMRT7 or During the evolution of DMRT8 several mutations occurred other DMRT proteins and thus prevent DNA binding. Regu- within the DM domain-encoding sequence. As a result, none of lation of DNA binding mediated by such types of dominant- the analyzed DMRT8 genes encodes a typical DM domain. negative regulators has been discovered for some members While Dmrt8 genes of cat, dog, cow, pig, and rabbit still encode of the basic helix–loop–helix, leucine-zipper, and home- a DM-like domain, a partial or complete loss of the DM domain odomain transcription factor families [41–43]. On the other has occurred in primates, mouse, and rat. hand, it is also possible that DMRT8 regulates the activity of other DMRT proteins indirectly by competition for coregulatory Do DMRT8 genes have DM domain-independent functions? factors.

Despite the lack of the DM domain, DMRT8 genes have been Male-biased expression of X chromosomal Dmrt8 genes in the maintained on the X chromosome in the placental mammalian mouse lineage. Thus, DMRT8 proteins might exhibit a DM domain- independent function. Other functional domains located In human and mouse, conflicting results have been reported downstream of the DM domain might be present and important about the presence of male-biased genes on the X chromosome for DMRT8. In vertebrates, not much is known about the [44,45]. On the one hand, the X chromosome appears to be presence of other functional domains in DMRT proteins, as enriched for male-biased genes, consistent with Rice’s there is only little conservation outside the DM domain [2].In hypothesis [46], which states that male-biased genes advanta- Drosophila DSX, the carboxy-terminal-located domains are geous for male reproduction preferentially accumulate on the X important for protein dimerization and recruitment of coregu- chromosome. However, recent analysis of the genomic latory factors [39,40]. Regions of high sequence similarity are distribution of sex-biased genes in mouse revealed that the present in the carboxy-terminus of DMRT7 and DMRT8 (Fig. occurrence of X-linked male-biased genes depends on their 1a). It would be of special interest to investigate whether these temporal and spatial expression [45,47]. The X chromosome A.-M. Veith et al. / Genomics 88 (2006) 185–195 193 accumulates male-biased genes expressed in male germ (http://genome.uscs.edu/). DMRT8 exon/intron boundaries were identified by cells prior to MSCI (meiotic sex chromosome inactivation) computer-assisted comparison between cDNA, putative proteins, and genomic DNA using sim4 [53] (http://bioinformatics.ube.ca/recources/tools) and Wise2 and genes expressed in testicular somatic cells, but there is a [54] (http:/www.ebi.ac.uk/wise2/). lack of male-biased genes expressed in male germ cells during later stages of spermatogenesis [45,47,48]. During MSCI, Mice which occurs in both sexes shortly after birth, sex chromosomes are transcriptionally inactive in germ cells, but not in somatic Mice used in this study were from an Mf1 background. Embryos were cells. Therefore, it has been suggested that male-biased genes collected from timed matings, with noon of the day on which the mating plug expressed in male germ cells that underlie MSCI are eliminated was observed designated as E0.5. Presence or absence of the Y chromosome was analyzed by PCR using Zfy-specific primers [55]. rapidly from the sex chromosomes, and genes with important functions in these cells are located primarily on autosomes Analysis of Dmrt8 expression in embryonic and adult mice [45,47]. Dmrt8.1 and Dmrt8.2 in mouse show a male-biased ex- For RT-PCR analysis several organs from adult male and female mice were pression in mature testis. Dmrt8.1 is expressed in the somatic dissected. Total RNA was isolated from up to 100 mg tissue using PeqGold Sertoli cells, suggesting that it might be involved in some TriFast (PeqLab). For qRT-PCR analysis, urogenital ridge regions from single E8.5 and E9.5 embryos, single pairs of urogenital ridges (mesonephros and Sertoli cell-mediated aspects of testicular function. Dmrt8.2 gonad) from E10.5 and E11.5 embryos, single pairs of gonads from E12.5 to shows a dynamic expression during embryogenesis exclusively E15.5 embryos, single gonads of 1-day to 5-week-old mice, and various organs in male and female gonads, with a peak at E13.5 in males. Sry, from single E13.5 embryos were prepared. Total RNA was isolated using the the sex-determining gene in mouse, is expressed in males from Absolutely RNA Microprep Kit (Stratagene). Reverse transcription and qRT- E10.5 to E12.5 with a peak at E11.5 [49]. Thus, the time course PCR analysis were performed as described by Barrionuevo and colleagues [56]. The relative expression in developing gonads was determined by comparison to of Dmrt8.2 expression suggests a potential role of Dmrt8.2 a serial dilution of adult testis cDNA using the iCycler IQ optical system during male sex differentiation downstream of Sry. After birth, software version 3.1. Relative Dmrt8 expression levels determined by this expression of Dmrt8.2 becomes restricted to testis, suggesting method were normalized to the corresponding amounts of Tbp. The following that Dmrt8.2 is expressed in somatic cells rather than in germ primers were used for Tbp: Tbp–f(5′-GCCAAGCCCTGAGCATAA-3′), Tbp-r ′ ′ cells of the testis as the sex chromosomes have been silenced by (5 -GGCCTCTCAGAAGCATCACTA-3 ), 55°C for 25 cycles. For qRT-PCR the following primers were used: Dmrt8.1-f (5′-AAGAGACGCCTGGTTGA- MSCI. The expression patterns of Dmrt8 genes in mouse are GAG-3′), Dmrt8.1-r (5′-GTTGTTTCTGGGTGCTGGTT-3′), Dmrt8.2-f (5′- consistent with the hypothesis of a preferential accumulation of TTTCACAAGCATGGCAAGAG-3′), Dmrt8.2-r (5′-TCACTGGAGCATA- male-biased genes on the X chromosome. From there, DM AGGCAGA-3′), 60°C for 40 cycles. Primers used for RT-PCR were, for domain-less X-linked Dmrt8 genes in mouse might exhibit Dmrt8.1a, Dmrt8.1-f1 (5′-actgaaaatgcagcggtctt-3′), Dmrt8.1-r1 (5′-catacgt- ′ ′ some functions advantageous in males. gatgctccgctta-3 ), 55°C for 35 cycles; for Dmrt8.1b and c, Dmrt8.1-f3 (5 - atggtggaggaggaagtagc-3′), Dmrt8.1-r3 (5′-ttaagtcaacagggacatgc-3′), 55°C for Our observations support the involvement of several DMRT 40 cycles; for Dmrt8.2a, Dmrt8.2-f1 (5′-AGCGGTCTTCGCGTTCCT-3′), genes other than Dmrt1 during sexual development in the Dmrt8.2-r3 (5′-GTGTTCCATGTCCAGGAAG-3′), 55°C for 35 cycles; and mouse [30]. Dmrt1 is expressed at similar levels in gonads of for Dmrt8.2b and c, Dmrt8.2-f4 (5′-atgacaatttctacaaccat-3′), Dmrt8.2-r3, 55·C male and female mouse embryos from E11.5 to E13.5 [16,50]. for 40 cycles. At E14.5, Dmrt1 expression becomes male-specific and in adults it is expressed in Sertoli cells and germ cells [16,17]. Cloning and sequencing of DMRT8 PCR products Dmrt4 is detectable in gonads of both sexes from E11.5 on. In Dmrt8 RT-PCR products derived from testis cDNA were cloned into embryos at E15.5, Dmrt3 is expressed mainly in interstitial pBluescript II KS(+) (Stratagene) or into the pCRII TOPO vector (Invitrogen cells. Dmrt7 expression is detected at higher levels in the Life Technologies). Sequencing reactions were performed using the CEQ DTCS developing ovary than in testis, suggesting a role for Dmrt7 dye terminator cycle sequencing kit and run on a CEQ 8000 DNA sequencing during ovary differentiation in mouse [30]. One function of the system (Beckman Coulter). Dmrt8 proteins in the mouse could be the direct or indirect regulation of Dmrt7 or other DMRT proteins, possibly as RNA in situ hybridization dominant negative regulators and thus to promote male sexual In situ hybridizations were performed on paraffin sections of adult testis development. using digoxigenin-labeled (Roche) antisense and sense riboprobes [57]. For Dmrt8.1 a 610-bp cDNA fragment and for Dmrt8.2 a 609-bp cDNA fragment Materials and methods from testis, subcloned into pBluescript II KS(+), were used to generate riboprobes. A Wt1-specific probe was generated from a 1084-bp fragment Sequence analysis and database screening containing the 3′ end of Wt1 mRNA.

Sequence analysis and multiple sequence alignments were performed using BioEdit (sequence alignment editor, 1997–2004, Tom Hall, Isis Pharmaceu- Acknowledgments ticals, http://www.mbio.ncsu/BioEdit) and implemented modules, like ClustalX [51]. Phylogenetic analysis was performed on single and combined amino acid This work was supported by the BioFuture program of the alignments of regions I (75 aa), II (52 aa), III (35 aa), and IV (48 aa), with the German Bundesministerium für Bildung und Forschung (to J.N. neighbor-joining method [52], 1000 pseudosamples as implemented in PAUP* (D.L. Swofford, Sinauer Associates, Sunderland, MA, USA, 1998). BLAST V.) and by Grant En 280/6-1 from the Deutsche Forschungsge- analysis was done using sequence databases accessible from the NCBI server meinschaft (to C.E.). A.D. is a recipient of a postdoctoral (http://www.ncbi.nml.nih.gov/BLAST) and from the UCSC Genome Browser fellowship from the Alexander von Humbold Foundation. We 194 A.-M. 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