Short report

Developmentally regulated and non-sex-specific expression of autosomal

dmrt genes in embryos of the Medaka fish (Oryzias latipes)

Christoph Winklera,*, Ute Hornunga, Mariko Kondoa,1, Cordula Neunera, Jutta Duschla,Akihiro Shimab, Manfred Schartla

aDepartment of Physiological Chemistry I, Biocenter, University of Wuerzburg, Wuerzburg, GermanybDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo, Japan

Received 7 July 2003; received in revised form 23 March 2004; accepted 24 March 2004

Abstract

The dmrt2gene family of vertebrates comprises several transcription factors that share a highly conserved DNA-binding domain, the DM

domain. Like some of their invertebrate counterparts, e.g. Drosophila doublesex (dsx) and the Caenorhabditis elegans Mab3, several are

implicated in sex determination and differentiation. Thus far, dmrt genes represent the only factors involved in sexual development that are

conserved across phyla. In the teleost Medaka (Oryzias latipes), a duplicated copy of dmrt1, designated dmrt1bY or dmy, has recently been

postulated to be the master regulator of male development in this species. Here, we have analyzed the expression of four additional Medaka

dmrt genes during embryonic and larval development. In contrast to other vertebrates, the autosomally located dmrt1a gene of Medaka is not

expressed at detectable levels during embryogenesis. On the other hand, dmrt2, dmrt3 and dmrt4 show highly restricted and non-overlapping

expression patterns during embryogenesis. While dmrt2 is expressed in early somites, dmrt3 transcripts are found in dorsal interneurons and

dmrt4 is expressed in the developing olfactory system. Other than in mouse, they do not show any sex specific expression and no transcription

could be detected in the early developing gonads. However, all four analyzed dmrt genes share expression in the differentiating gonad of

larvae and in adult testis.

q 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: DM domain genes; DMRT; Sex determination; Sex differentiation; Somitogenesis; Olfactory system

1. Results and discussion

The dmrt genes have attracted considerable interest

recently because of their involvement in sex determination

and differentiation across invertebrate and vertebrate

species. The first known and prototype member, dmrt1, is

implicated in vertebrate male development, although with

some species-specific differences. In humans, haploin-

sufficency of dmrt1 leads to male to female sex reversal

(Raymond et al., 1999a) and knockout mice have severe

defects in testis differentiation (Raymond et al., 2000).

Thus, in mammals dmrt1 is thought to act as a sex

determination/differentiation gene occupying a more down-

stream position in the genetic cascade directing

the development of the gonad. In chicken, dmrt1 is located

on the Z chromosome (Nanda et al., 2000). Its expression

pattern is consistent with being an excellent candidate for

the master male sex-determining gene of birds (Smith et al.,

1999; Raymond et al., 1999b). In the turtle, where sex is

determined by the ambient temperature, dmrt1 expression is

higher in the gonads of embryos incubated at the male

determining temperature preceding sexual differentiation

(Kettlewell et al., 2000). In the fish Medaka, a duplicated,

additional copy of dmrt1, denominated dmrt1bY or dmy,

resides at the male sex determining locus on the Y

chromosome and shows all features of the master sex

determining gene (Matsuda et al., 2002; Nanda et al., 2002).

However, in other fish species investigated no homologue of

dmrt1bY was detectable (Kondo et al., 2003), making it

unlikely that this gene is the male sex determining gene in

most fish (Volff et al., 2003a). dmrt1 expression precedes

0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mod.2004.03.018

Mechanisms of Development 121 (2004) 997–1005

www.elsevier.com/locate/modo

1 Present address: Department of Biological Sciences, University of

Tokyo, Tokyo, Japan.2 For simplicity, the fish terminology has been used for all vertebrate dmrt

genes throughout the manuscript (for details see Volff et al., 2003b).

* Corresponding author. Tel.: þ49-931-888-4142; fax: þ49-931-888-

4150.

E-mail address: cwinkler@biozentrum.uni-wuerzburg.de (C. Winkler).

testes development in the rainbow trout (Marchand et al.,

2000) and in adults is exclusively expressed in testes of

fugu, trout, Medaka and Xiphophorus (Marchand et al.,

2000, Brunner et al., 2001; Veith et al., 2003).

Several other dmrt genes also show a gonadal expression:

in the mouse embryo, dmrt3 is expressed in the interstitial

cells of the developing testis, dmrt4 is found in gonads of

both sexes and dmrt7 is expressed in the developing ovary.

This pattern persists in the adult gonads, where additionally

dmrt5 and 2 (preferentially in ovary) are expressed (Kim

et al., 2003). In the fish Medaka, preferential expression of

dmrt3 in testes has been reported, while dmrt2 and 4

appeared equally expressed in the male and female gonad,

as analyzed by RT-PCR (Brunner et al., 2001). There is

even evidence for species-specificity of expression. The

dmrt4 was described in tilapia as an ovary specific gene

(Guan et al., 2000) while it is expressed in Medaka in testes

and many other organs as well (Kondo et al., 2002).

Another peculiarity of the dmrt gene family concerns the

genomic organization. dmrt1–3 form a cluster on human

chromosome 9p24.3. This cluster organization and linkage

to some other neighboring genes is conserved from man to

fish. dmrt5 and 6 are clustered in the mouse and human

genome as well (Kim et al., 2003). This has lent support to

the idea that a common expression in gonads is connected to

this organization (Ottolenghi et al., 2000), reminiscent of

the hox cluster.

In order to understand the function of the dmrt genes for

vertebrate development, a first step is to differentiate

between the conserved functions and those that diverged

during the evolution of vertebrates. One approach is to

compare the embryonic expression patterns from different

vertebrate model species. Comprehensive analyses of dmrt

gene expression in the mouse and chick embryo are now

available (Kim et al., 2003; Smith et al., 2003). We have

performed a similar study in the Medaka (Oryzias latipes)

for dmrt1–4. Orthologues for dmrt6 and 7 could not been

identified in fish, including the draft version of the sequence

of the genome of fugu (Volff et al., 2003b). It is a well

known fact that teleost fish have additional paralogs of

many gene families, either due to whole genome duplication

or tandem gene duplications (Amores et al., 1998; Wittbrodt

et al., 1998). In Medaka, at present no duplicates other than

for dmrt1 (Nanda et al., 2002) and dmrt2 (A. Veith and J.N.

Volff, personal communication) have been identified.

To analyze the temporal expression of different Medaka

dmrt genes during the very early phase of embryogenesis,

RT-PCR was performed on cDNA prepared from pooled

female and male embryos of the Medaka quart strain at

different stages of early development (Fig. 1A). As reported

earlier for Medaka of the Carbio strain, no expression of

dmrt1a (the Medaka orthologue of the dmrt1 gene has the

suffix a to distinguish it from its coorthologue on the Y

chromosome) could be detected from day 1 to 7 of

development, while strong expression in testis was observed

(Fig. 1B, right lane). For dmrt2, weak transcription was

detected on day 1 of development that increased on day 2

and persisted throughout early development. Interestingly,

dmrt3 showed a transient mode of transcription with a peak

of expression first detectable on day 4 of development. In

contrast to this, dmrt4 was already strongly expressed on

day 1. Transcription decreased on day 3 but reoccurred

towards the end of embryogenesis. We next wanted to know

whether dmrt genes show any differences in expression in

the two sexes. For this, again embryos and larvae of the

quart strain were used, where sexes can be distinguished

due to expression of a sex chromosome linked pigmentation

marker as early as from day 4 of embryonic development

(Wada et al., 1998). Unlike the Y-chromosomal dmrt1bY

gene, which is male specifically expressed during embryo-

nic and larval development (Nanda et al., 2002), no sex

specific difference was observed for the other dmrt genes.

dmrt1a expression was undetectable consistent with earlier

reports (Nanda et al., 2002). Taken together, all four

analyzed dmrt genes show different temporal expression

profiles during early development and exhibit no sex-

specific differences with respect to their expression patterns.

For analyzing the spatial expression, RNA in situ

hybridization studies were performed at different stages of

development. Consistent with the RT-PCR results, we could

not detect any expression of dmrt1a during embryogenesis

(Fig. 1C) while in comparison expression of vasa was

readily detectable in the developing gonad from early stages

onwards (Fig. 1D,E). However, whole mount staining of

adult testis using the same riboprobe revealed strong

expression of dmrt1a (Fig. 2B). Histological analysis

showed expression of dmrt1a in Sertoli cells, characterized

by their typical morphology and peripheral location in cysts,

but no expression in the surrounding spherical germ cell

precursors (Fig. 2C,D). Also dmrt2–4 showed strong

expression in adult testis (Fig. 2F–H) confirming the results

obtained by RT-PCR (Brunner et al., 2001; Kondo et al.,

2002). As shown earlier by RT-PCR (Ohmuro-Matsuyama

et al., 2003; and our unpublished data), our in situ analysis

also identified weak dmrt1a expression in the ovary. There,

transcripts were detected in the early differentiating oocytes

(Fig. 2I). Also dmrt2 showed expression in the ovary, which

was restricted to early stage oocytes, at significantly higher

levels compared to the other dmrt genes (Fig. 2J). In

accordance with RT-PCR data (Brunner et al., 2001), no

expression of dmrt3 was observed in ovary. dmrt4 was weak

and only visible in primary oocytes. We next analyzed dmrt

expression in the differentiating gonads of Medaka larvae at

12 and 20 days post hatching (dph). The gonadal region

again was visualized by analysis of vasa expression in germ

cell precursors (Fig. 2M,T). Other than during embryonic

gonad development, dmrt1a showed strong expression at 12

dph in the vasa positive, bilateral gonad, located directly

dorsal to the intestine (Fig. 2N,S). Also, dmrt2 and dmrt3

are expressed in this region at 12 dph (Fig. 2O,P). In

contrast, dmrt4 expression could not be detected at 12 dph,

but was evident at 20 dph (Fig. 2Q,R). Taken together, all

C. Winkler et al. / Mechanisms of Development 121 (2004) 997–1005998

analyzed dmrt genes share strong expression in the adult

testis and the differentiating gonad of Medaka larvae, but

show differential regulation in the adult ovary.

During embryonic development, all analyzed dmrt

genes, except dmrt1a, show strong and highly restricted

expression. For dmrt2, expression could be detected from

earliest somitogenesis stages onwards (Fig. 3A–D). At one

day post fertilization (1 dpf), expression was confined to

the already formed somites and the most anterior region of

the presomitic mesoderm (PSM). The region stained in the

PSM represents the next somite to be formed (somitomere

S0, arrowhead in Fig. 3C,D). In the epithelialized somites,

expression levels appeared slightly elevated in the caudal

portions. At later stages of development (4 days post

fertilization) expression is found in all somites throughout

the trunk region, in the mesoderm of the tail, the

pharyngial arches and in the brain (Fig. 3E and data not

shown). In the somites, expression is stronger in the caudal

halves of the ventral domains that will later give rise to

the sclerotome (reviewed by Stickney et al., 2000), but low

level of expression are also found dorsally (Fig. 3F).

Consistent with RT-PCR data, overall transcription levels

decreased at 6 days of development when transcription is

hardly detectable in the dorsal somite domain (Fig. 3G).

The dmrt2 expression in Medaka is similar to that in the

mouse and zebrafish, where it also is expressed in the

somites and anteriormost PSM (Meng et al., 1999; Kim

et al., 2003). However, Medaka dmrt2 is also expressed in

the developing head, while both the mouse and zebrafish

orthologs are not.

In contrast to the other paralogs, dmrt3 shows a very

transient mode of expression. No transcripts were detectable

at and before stage 26 (Fig. 4A) and only low level

expression was observed at stages later than stg. 36

(Fig. 4B). In between, strong expression was found in two

parallel rows of cells spanning the complete anteroposterior

axis of the hindbrain and the neural tube (Fig. 4C,D).

Transcripts were found in small clusters of directly adjacent

Fig. 1. (A,B) Expression of dmrt genes during embryonic and larval development of male and female Medaka (quart strain) as analyzed by RT-PCR. For early

development (A) male and female embryos had to be pooled, for later stages (B) they were separated according to their sex-specific pigmentation patterns. No

cDNA was added to the PCR in the negative control. For positive control, cDNA from testes of an adult male (Carbio strain) was used. (C) Lateral view of

Medaka embryo at 4 days post fertilization (dpf; stage 31) showing no specific staining after using a dmrt1a antisense riboprobe. (D) Lateral view of embryo at

2.3 dpf (stg. 26) with strong vasa expression in the primordial germ cells of the early gonad region. (E) Higher magnification image of vasa positive cells in the

gonadal region in a different embryo of the same stage.

C. Winkler et al. / Mechanisms of Development 121 (2004) 997–1005 999

cells (Fig. 4E) and histological analysis revealed that these

clusters were located at mediolateral positions inside the

neural tube (Fig. 4F). From this position, the dmrt3 positive

cells are likely to represent a subset of differentiating dorsal

interneurons. Identical expression patterns have been

reported for the dmrt3 orthologs in mouse and chicken

(Kim et al., 2003; Smith et al., 2003).

While dmrt2 is expressed in mesodermally derived

tissues and dmrt3 is restricted to the neural tube, dmrt4 is

expressed in the ectodermally derived olfactory placode and

the neuroectodermal telencephalon (Fig. 5). Low-level

transcription was also found in the otic placodes (data not

shown). Expression of dmrt4 in the telencephalon is

dynamic. While transcripts are distributed throughout

Fig. 2. Expression of dmrt genes in adult and developing gonads as revealed by whole mount RNA in situ hybridization. (A–D) Expression of dmrt1a in adult

testis. (A) Sense control. (B) Detection of dmrt1a using an antisense probe. (C) Section of whole mount stained testis counterstained with Eosin showing

expression of dmrt1a (in blue; marked by arrows) in Sertoli cells. (D) Higher magnification view of (C) showing morphology of Sertoli cells and characteristic

position in the periphery of testis cysts. (E–H) Expression of vasa (E), dmrt2 (F), dmrt3 (G) and dmrt4 (H) in adult testis. (I–L) Expression of dmrt1a (I), dmrt2

(J), dmrt3 (K) and dmrt4 (L) in adult ovaries. Weak expression of dmrt1a and strong expression of dmrt2 in primary oocytes is indicated by arrows (I,J). (M–T)

Expression of vasa (M,T) and dmrt genes (N–S) in the gonadal regions of Medaka larvae at 12 (M–Q, S) and 20 (R,T) days post hatching (dph). Lateral views

of trunk regions in (M)–(R), the anus is marked with asterisks. Note absence of dmrt4 expression at 12 dph (arrow in Q), but elevated levels of expression at 20

dph (arrows in R). In (R) the gonad attached to the intestine has been partially dissected from the peritoneum. (S,T) Ventral views of trunk regions showing

bilateral expression of dmrt1a at 12 dph (S) and vasa at 20 dph (T). Anterior is to the left in (M)–(T).

C. Winkler et al. / Mechanisms of Development 121 (2004) 997–10051000

Fig. 3. Expression of dmrt2 during somitogenesis. (A) Dorsal view of flat-mounted embryo at stage 21, which corresponds to approximately 34 h post

fertilization (hpf) at an incubation temperature of 26 8C. Arrows mark dmrt2 expression in the first forming somites. (B,C) Lateral view of same embryo at low

(B) and high (C) magnification showing expression in the first five epithelialized somites, as well as the next emerging somite (somite S0; marked by

arrowhead). (D) Close-up dorsal view of same embryo showing that the S0 somite expresses dmrt2 in its posterior domain while the presomitic mesoderm

(PSM) lacks expression. E. Lateral view of a stage 31 embryo (corresponding to 4 dpf) showing dmrt2 expression in the somites and the head. (F) Close-up

lateral view of the trunk region of the same embryo as in (E) with anterior to the left. Boundaries of one somite are marked by red dashed lines. Red arrows

indicate elevated dmrt2 expression in the ventral and caudal domains of the somites. (G) At stg. 36 (6 dpf), expression levels in the trunk somites are reduced.

e, eye; mb, midbrain.

C. Winkler et al. / Mechanisms of Development 121 (2004) 997–1005 1001

the forebrain at early stages (stg. 22; Fig. 5A), the

expression domain becomes more and more restricted to a

small area around the first ventricle in the dorsal

telencephalon at stg. 26 (Fig. 5B,C). At later stages, hardly

any transcripts are present in the forebrain, but strong

expression can be seen in the developing nose (Fig. 5D).

Taken together, while all members of the dmrt gene

family in Medaka analyzed in this work share an expression

in the male gonad, they show restricted and significantly

different and non-overlapping patterns during embryonic

development. This situation is similar to that reported in

higher vertebrates. Many of the orthologs show similarities

Fig. 4. Transient expression of dmrt3 in the developing neural tube. (A,B) Lateral views of embryos at stage 26 (A) and 36 (B) showing no (A) or low (B) levels

of dmrt3 transcription. (C,D) In contrast to this, high expression levels are seen at stage 31 (corresponding to 4 dpf). The dorsal view in (C) shows two parallel

rows of dmrt3 positive cells in the hindbrain and spinal cord. In a lateral view (D) the position of these cells is medial in the neural tube. (E) High magnification

lateral view of embryo at the same stage showing trunk region with segmental clusters of dmrt3 positive cells. Neural tube (nt) and notochord (nc) are indicated.

(F) Cross section through the trunk region of an embryo at stg. 31 showing mediolateral position of the dmrt3 positive cell clusters in the neural tube

(nc, notochord).

C. Winkler et al. / Mechanisms of Development 121 (2004) 997–10051002

in expression patterns in different species. Intriguingly,

however, there are also significant differences. For example,

while in both mouse and chick dmrt3 is expressed in the

telencephalon and nasal pits (Smith et al., 2003), in Medaka

dmrt4 is expressed in these tissues. Yet, the dmrt3 orthologs

are identically expressed in the neural tube in mouse, chick

and Medaka. Interestingly, however, the chicken dmrt3

gene is also expressed in the PSM where it is not expressed

in both mouse (Smith et al., 2003) and Medaka. In Medaka,

the only dmrt gene analyzed so far that is expressed in the

anterior PSM is dmrt2. The spatial and temporal regulation

in PSM and somites appears identical in all three species,

with, e.g. reduced transcription in aging somites. Finally,

while none of the analyzed dmrt genes in Medaka is

expressed in the early developing gonad, expression of some

of the corresponding mouse orthologs (dmrt1–4) has been

described in this organ (Kim et al., 2003). Therefore, while

some functional redundancy of different dmrt genes in the

mouse is thought to be responsible for sexual differentiation,

this might not be the case in the Medaka. In summary, this

indicates species-specific evolution of gene expression

patterns with particular domains representative for one

paralog in one species, but on the other hand characteristic

for other paralogs in distantly related species.

Due to its role in sex determination, dmrt1 is the most

prominent member of this family. However, the plasticity of

dmrt1 function is not understood at all. Interestingly, the

invertebrate homologues of dmrt1, the Drosophila double-

sex (dsx) and the Caenorhabditis elegans mab-3 genes, are

well-defined regulators of sexual development albeit at a

downstream position of the genetic cascade (for review see

Zarkower, 2001). Notably this makes dmrt1 the single sex

regulatory gene so far that is conserved between different

phyla, while other pathway components are highly variable

(Raymond et al., 1998). This situation is markedly different

from most other developmental pathways where many

components are highly conserved.

2. Experimental procedures

2.1. RT-PCR

Total RNA was extracted from 10–20 pooled total male

and female embryos using the TRIZOL reagent (Gibco-

BRL) according to the supplier’s recommendation. After

DNAse treatment reverse transcription was done with 4 mg

total RNA using Superscript II reverse transcriptase (Gibco-

BRL) and random primers. cDNA from 10 ng (actin) or

600 ng (dmrt’s) of total RNA was used for PCR with gene

specific primers (Table 1).

2.2. In situ hybridization

Whole-mount RNA in situ hybridization was performed

as described (Winkler and Moon, 2001). Stained embryos

were manually sectioned and mounted in glycerol for

photography. Pieces of testis and ovary tissue were stained

with the same protocol as embryos with a proteinase K

incubation of two minutes. Stained testes were embedded in

paraffin, sectioned and counterstained with Eosin. For in situ

analysis of trunk regions from Medaka larvae, heads and

tails were removed after fixation. Proteinase K was used at a

concentration of 20 mg/ml and the incubation time was

increased to 6 h at room temperature. The dmrt1a probe for

in situ hybridization was obtained by cloning the full-length

cDNA into pCS2þ . The resulting plasmid pCSdmrt1a was

linearized with BamHI and antisense RNA was prepared

using the T7 RNA polymerase.

Fig. 5. Expression of dmrt4 in the developing olfactory system. (A) Dorsal

view of flat-mounted Medaka embryo at stg. 22 (1.5 dpf) showing dmrt4

expression in the early olfactory placodes (OP) and throughout the

forebrain (FB; R, retina). (B,C) Dorsal (B) and frontal (C) view of anterior

head region of an embryo at stg. 26 (2.3 dpf) showing strong dmrt4

expression in the olfactory placodes and restricted expression in the dorsal

telencephalon (TC). (D) Dorsal view at stg. 33 (4.5 dpf) with dmrt4

expression in the nasal pits. dmrt4 transcription in the telencephalon is no

longer detectable.

C. Winkler et al. / Mechanisms of Development 121 (2004) 997–1005 1003

Medaka dmrt2 was amplified from embryonic cDNA

using the primers DMRT2-06 (50-GCTCCTTGCA-

GAAGGGTGTT-30) and DMRT2a (see Table 1). The

amplified 1.17 kb fragment was cloned into the Sma I site

of Bluescript KS2þ . For preparation of RNA antisense

probes, this plasmid was linearized with Not I, and the T3

RNA polymerase was used for in vitro transcription.

The Medaka dmrt3 riboprobe contained the full-length

coding region of dmrt3 and was obtained by RT-PCR using

the primers DMT3a (50-ATGAACGGCTACGGCTCTCC-30)

and DMT3b (50-TCACGCGGAGTCGGAGCGC-30). The

amplified 1.33 kb fragment was cloned into the Sma I site of

pBluescript KS2 þ and riboprobes were obtained by

digestion with Xho I and transcription with the T7 RNA

polymerase.

For the dmrt4 riboprobe, a 363 bp fragment was PCR

amplified from Medaka cDNA using the primers DMT4c

and DMT4d (see Table 1). This fragment was cloned into

the Sma I site of pBluescript KS2 þ and the resulting vector

linearized with Not I. Antisense transcripts were obtained

using the T3 RNA polymerase.

2.3. Fish

All Medaka were taken from closed colony breeding

stocks of the Carbio (Carolina Biological Supplies) and

quart (Wada et al., 1998) strains, which are kept under

standard conditions. The embryos were staged according to

Iwamatsu (1994).

Acknowledgements

We thank Jean-Nicolas Volff for critical comments on

the manuscript and Y. Nakahama and M. Tanaka for the

Medaka vasa (Olvas) riboprobe. This work was supported

by grants to M.S. supplied by the European Community

(FAIR CT 97-3796) and Fonds der Chemischen Industrie

and by the DLR (50 WB 0152) to C.W. and M.S.

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Table 1

Primers used for RT-PCR expression analysis

Gene Primer Sequence Tm (8C) Annealing

temperature (8C)

Product

size (bp)

Cycles

dmrt1a DMT1m TCC GGC TCC ACA GCG GTC 62.8 64 329 35

DMT1n CAG ACA GAG GGT TGG GGG G 63.1

dmrt2 DMT2a GCG ACC AGC GGA AGC TGA G 63.1 62 292 35

DMT2b CCT TCC AGG ATG CTT TTG GC 59.4

dmrt3 DMT4a CCC CGC TGC AGC GGA CCC 67.4 66 200 35

DMT4b GAT GAG GCT CTC CAG ACT CTC 61.8

dmrt4 DMT4c GCG GGA CCT GCG CCT TCT G 65.3 66 363 35

DMT4d GCG GGC CTC TGC GGG CTC 67.4

actin MAct1 TTC AAC AGC CCT GCC ATG TA 57.3 60 349 25

MAct2 GCA GCT CAT AGC TCT TCT CCA GGG AG 68.0

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