Developmental Brain Research 148 (2004) 223 – 232
www.elsevier.com/locate/devbrainres

Gene Expression Pattern

Expression of DDAH1 in chick and rat embryos
Takuya Mishima a, Tsuyoshi Hamada b, Kumiko Ui-Tei c, Fumitaka Takahashi c, Yuhei Miyata d,Junko Imaki a,*, Hidenori Suzuki d, Kazuo Yamashita abaDepartment of Anatomy, Nippon Medical School, Bunkyo, Tokyo 113-8602, Japan
Creative Research Promoting Program Stem Cell Translational Laboratory, Riken Kobe Institute Center for Developmental Biology, Chuo,
Kobe 650-0047, Japanc
UPBSB, Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo, Tokyo 113-0033, Japand
Department of Pharmacology, Nippon Medical School, Bunkyo, Tokyo 113-8602, Japan

Accepted 10 September 2003

Abstract
Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is an enzyme that metabolizes methylated arginine to citrulline andmethylamine, thus working to produce nitric oxide (NO). We isolated a gene encoding chick DDAH1. In situ hybridization analysis
revealed characteristic DDAH1 mRNA expression in the embryonic spinal cord, which was especially strong in the ventral horn and dorsal
root ganglion (DRG). DDAH1 was also detected in the brain, kidney, digestive tract, and in other tissues. We examined the expression
pattern of DDAH1 in developing rats and compared this with the expression pattern in chicks. The expression pattern in the rats was very
similar to that in the chicks, but there were some differences between the chicks and rats in the amount of DDAH1 detected in the heart,
liver, lung, and DRG. We also investigated neural nitric oxide synthase (nNOS) mRNA expression patterns in rat embryos. The DDAH1
expression patterns were completely different from nNOS expression patterns. Our study suggests that DDAH1 plays an important role indevelopment.
D 2004 Elsevier B.V. All Rights Reserved.
Keywords: Central nervous system; Chick; DDAH; Development; Dorsal root ganglion; Esophagus; Heart; In situ hybridization; Intestine; Kidney; Liver;
Lung; Rat; Rectum; Retina; Spinal cord; Stomach

1. Results

ogous to human DDAH1 (85%) and rat DDAH1 (84%)(Fig. 1).

1.1. Cloning of chick DDAH1
1.2. Expression of DDAH1 mRNA in CNS
To identify proteins involved in spinal cord differentiation, we screened for genes, whose expression in chickspinal cords changed between embryonic days 4 (E4) and10 (E10) using the differential display method, andobtained several cDNA fragments. We selected one of
the fragments that showed increasing expression between
E4 and E10. Using this fragment as a probe, we isolated
full-length cDNA of the gene from the E5 chick cDNA
library and sequenced it. This sequence encodes a putative protein composed of 287 amino acids and is homol-* Corresponding author. Tel.: +81-3-3822-2131; fax: +81-3-56853052.
E-mail address: jimaki@nms.ac.jp (J. Imaki).
0165-3806/$ - see front matter D 2004 Elsevier B.V. All Rights Reserved.
doi:10.1016/j.devbrainres.2003.09.021

1.2.1. Expression in the spinal cords
We examined chicks on days E4, E6, E8, E10, E14 andE18 (Figs. 2A –O and 3) and rats on days E14 and E17(Figs. 2P – U and 4). Signals were detected in the marginallayer of the spinal cord of the E4 chicks (Figs. 2A – C and
3A – C) and signal intension was found to be in the order ofcervical, thoracic and lumbar spinal cord. At chick stage E6,
signals were also detected in the marginal layer, especiallyin the ventral portion, and were first detected in the dorsal
root ganglion (DRG) and sympathetic ganglion (SG) (Figs.
2D – F and 3D – F). At chick stages E8 (Figs. 2G –I and 3G –I) and E10 (Figs. 2J– L and 3J– L), the expression pattern
was almost identical to that of E6. At E14 (Fig. 2M) andE18 (Fig. 2N,O) in the chicks, the signals became gradually

224

T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

Fig. 1. Comparison of amino acid sequences of DDAH1 genes. Accession numbers for DDAH1: chick, AB095027; human, NM_012137; rat, NM_022297.

weaker in the gray matter, but were still rather strong in
DRG and SG. In contrast, in the rats on both E14 (Figs. 2P –R and 4A – F) and E17 (Figs. 2S– U and 4G – J), signals
were detected in the gray matter, especially in the ventral
portion (probably coincident with motor neurons on E17),
and SG, but interestingly, were detected very weakly or not
at all in the DRG.
1.2.2. Expression in the rat brain
We examined the rat brains on E14 and E17 (Fig. 5). On
E14, signals were detected in the entire ventricular zone
(VZ) (Fig. 5A,B,D) and the tegmentum (Tg) (Fig. 5C). On
E17, signals were observed in the VZ (Fig. 5E –G,I,J), thechoroid plexus (ChP) (Fig. 5G –J), the ciliary ganglion (CG)(Fig. 5F), the inferior olive (IO) (Fig. 5I), the 7th cranial
nerve (7th) (Fig. 5H), the interpeduncular nucleus (IpN)(Fig. 5H), the red nucleus (RN) (Fig. 5H), the dorsal
tegmental nucleus (DTN) (Fig. 5I) and the nucleus of thelateral lemniscus (NLL) (Fig. 5H).
1.3. Expression of DDAH1 mRNA in thoracic organs
We examined thoracic organs in the chicks on E4, E6, E8
and E10 (Figs. 3B – E,G,H,J,K and 6A – E) and thoracic
organs in the rats on E14 and E17 (Figs. 4A,B,G,H and 6F –J). At chick stage E4, strong signals were observed in theaorta (Fig. 6A) and in the esophagus (Fig. 6B). Signals in

the aorta in the following stages gradually weakened (Fig.
6C– E). Signals in the esophagus were observed mainly inthe smooth muscle and in the mucosa on E4 (Fig. 6B) andE6 (Fig. 6C). In the smooth muscle, signals gradually
weakened in the following stages (Fig. 6D,E), while in themucosa, signals continued to be intense (Fig. 6D,E).
In the rats on E14 and E17, signals were also detected in
the aorta (Fig. 6F– H) and in the esophagus (Fig. 6F– H). In
the rat esophagus, signals in the mucosa were not as strongas those in the chick mucosa, but the signal patterns werevery similar. In the rat respiratory system (Fig. 6F – H) andheart ventricles (Fig. 6I,J), in contrast with the chickrespiratory system and heart ventricles, intense signals weredetected.
1.4. Expression of DDAH1 mRNA in abdominal organs
We examined abdominal organs in the chicks on E6, E8
and E10 (Figs. 3E,F,H,I,L and 7A – H) and abdominalorgans in the rats on E14 and E17 (Figs. 4B – F,I,J and
7I– P). In the chicks, strong signals were detected in the
tubules of the kidney (Fig. 7A – C), in the serosa of thestomach (Fig. 7D –F), in the serosa of and in the boundary
between the smooth muscle and submucosa of the intestine(Fig. 7G,H), and in the rectum expression patterns weresimilar to those for signals detected in the intestine (Fig.
7B,C). Moderate signals were detected in the mucosa andT. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

225

Fig. 2. In situ hybridization analyses of DDAH1 mRNA in the developing spinal cord of the chicks on days E4 (A – C), E6 (D – F), E8 (G – I), E10 (J – L), E14(M) and E18 (N, O), and of the rats on E14 (P – R) and E17 (S – U). These figures are at the cervical (A, D, G, J, M, N, P, S), thoracic (B, E, H, K, Q, T) or
lumbar (C, F, I, L, O, R, U) levels. CC, central canal; DMC, dorsolateral motor columns; DRG, dorsal root ganglion; EL, ependymal layer; GM, gray matter;
ML, marginal layer; SG, sympathetic ganglion; Ver, vertebrae; VMC, ventral motor columns; VlMC, ventrolateral motor columns; VmMC, ventromedial motor
columns; VR, ventral root; WM, white matter.

226

T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

Fig. 3. DDAH1 mRNA expression pattern (A – L) and adjacent Nissl-stained sections (a – l) in the chick bodies on E4 (A – C), E6 (D – F), E8 (G – I) and E10(J – L). Each section is at the cervical (A, D, G, J), thoracic (B, C, E, H, K) or lumbar (F, I, L) level. Ao, aorta; Atr, atrium; CA, carotid artery; Dien,diencephalon; DRG, dorsal root ganglion; Eso, esophagus; In, intestine; Ki, kidney; Li, liver; Lu, lung; Nc, notochord; Re, rectum; SC, spinal cord; SG,sympathetic ganglion; St, stomach; SVC, superior vena cava; Tele, telencephalon; Ven, ventricle.

T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

227

Fig. 4. DDAH1 mRNA expression pattern (A – J) and adjacent Nissl-stained sections (a – j) in the rats bodies on E14 (A – F) and E17 (G – J). Each section is at
the cervical (G), thoracic (A – C, H), or lumbar (D – F, I, J) level. Ao, aorta; Atr, atrium; Bla, bladder; CCA, common carotid artery; DRG, dorsal root ganglion;
Eso, esophagus; Hum, humerus; In, intestine; Ki, kidney; Li, liver; Lu, lung; MBr, main bronchus; Ova, ovary; Pan, pancreas; Re, rectum; SC, spinal cord; SG,sympathetic ganglion; St, stomach; Tra, trachea; Ven, ventricle.

228

T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

Fig. 5. In situ hybridization analyses of DDAH1 mRNA in rat brain coronal sections on E14 (A – D) and E17 (E – J). 7th, 7th cranial nerve; Bas, basioccipitalbone; CG, ciliary ganglion; ChP, choroid plexus; DTN, dorsal tegmental nucleus; IO, inferior olive; IpN, interpeduncular nucleus; NLL, nucleus of the lateral
lemniscus; NS, nasal septum; Ret, retina; RN, red nucleus; Tg, tegmentum; VZ, ventricular zone.

smooth muscle of the stomach (Fig. 7D –F), the intestine(Fig. 7G,H) and the rectum (Fig. 7B,C).
In the rats on E14 and E17, a similar expression pattern
to that found in the chicks was detected in the kidney (Fig.
7I,J), stomach (Fig. 7K,L), intestine (Fig. 7M) and rectum(Fig. 7N,O), but the signals were not as intense as thosefound in the chicks. Moreover, in the rats, signals weredetected in the liver (Fig. 7P), ovary (Fig. 7I), bladder (Fig.
7M,N), seminal vesicle (Fig. 7O) and pancreas (Fig. 7K,L).

detected mainly in the inner nuclear layer and intermediatezone (Fig. 9B). DDAH1 signal expression patterns werecompletely different from nNOS expression patterns (Fig.
9C). In other organs in rat embryos, DDAH1 expression
patterns were different from nNOS expression patterns (datanot shown).

1.5. Expression of DDAH1 mRNA in other organs

Differential display, molecular cloning and sequencing
were performed as described previously [2]. At each stageof Wistar rat and White leg-horn chick, embryos was fixedwith Zamboni’s fixative. Sections of 20 Am thickness were
cut on a cryostat and mounted onto silane-coated slides. In
situ hybridization was performed with 35S-UTP labeled
RNA riboprobes according to the method described by
Yoshida et al. [9]. The following primer pairs of DDAH1
were used, forward and backward, respectively: chick
DDAH1, AGCATCAGCTGTACGTG and TTTCTCCAGTTCTGTGTTGG, rat DDAH1, AGGCTGATGATGGCTCTGTA and ATCCAGAGTTCGAGACCTTG.

Signals were observed in the inner nuclear layer andpigment layer of the retina (Fig. 8A,B), in the salivary gland(Fig. 8C) and in the bones of both chick (Fig. 3I, see i Leg)and rat embryo (Fig. 8D –F).
1.6. Comparison of DDAH1 mRNA and neural nitric oxide
synthase (nNOS) mRNA in the brains of rats on E16
DDAH1 mRNA signals were detected mainly in the VZ
and DTN (Fig. 9A), and nNOS mRNA signals were

2. Materials and methods

T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

229

Fig. 6. In situ hybridization analyses of DDAH1 mRNA in the embryonic thoracic organs. A shows the chick aorta on E4. B – G show the mid-thoracic level inthe chicks on E4 (B), E6 (C), E8 (D) and E10 (E), and in the rat on E14 (F) and E17 (G), including lung, esophagus, aorta and bronchus. H is an upper thoracic
transverse section that includes the aortic arch, esophagus and trachea in the rats on E17. I and J show the rat hearts on E14 and E17, respectively. Ao, aorta;Atr, atrium; Eso, esophagus; LV, left ventricle; Lu, lung; MBr, main bronchus; RV, right ventricle; SC, spinal cord; SG, sympathetic ganglion; St, stomach; Tra,
trachea.

230
T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232
Fig. 7. In situ hybridization analyses of DDAH1 mRNA in the embryonic abdominal organs. A – C, I, J show the kidney and surrounding organs in the chicks on E6 (A), E8 (B) and E10 (C), and in the rats on E14(I) and E17 (J). D – F, K, L show the stomach and surrounding organs in the chicks on E6 (D), E8 (E) and E10 (F), and in the rats on E14 (K) and E17 (L). G, H, M – O show the enteral organs in the chicks on E8(G) and E10 (H), and in the rats on E14 (N) and E17 (M, O). P shows the rat abdominal aorta on E17. Ao, aorta; Bla, bladder; In, intestine; Ki, kidney; Li, liver; Ova, ovary; Pan, pancreas; Re, rectum; Sem,seminal vesicle; SG, sympathetic ganglion; St, stomach.

T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

231

Fig. 8. In situ hybridization analyses of DDAH1 mRNA in other embryonic organs. Signal expression in the eye of the chick on E6 (A) and of the rat on E17(B); expression in the salivary gland of the rat on E17 (C); expression in the bone of the rat on E14 (D) and E17 (E, F). Br, brain; Cil, ciliary muscle; EL, eyelid;
Fem, femur; HJ, hip joint; In, intestine; Inn, inner nuclear layer of retina; Ki, kidney; KJ, knee joint; Li, liver; Opt, optic nerve; Ova, ovary; Pig, pigment layerof retina; SGl, salivary gland; Tib, tibia; Ton, tongue.

Fig. 9. In situ hybridization analyses of DDAH1 mRNA in the rat E16 brain section (A) and nNOS mRNA in an adjacent section (B). C is digitallysynchronized figures A (DDAH1 signals (blue)) and B (nNOS signals (red)). IN, inner nuclear layer; IZ, intermediate zone; VZ, ventricular zone.

232

T. Mishima et al. / Developmental Brain Research 148 (2004) 223–232

A PstI fragment of rat nNOS cDNA (gift by Drs. Hirose andHagiwara, Tokyo Institute of Technology) was subcloned
into p-Bluescript and linealized with BamHI. Radioactive
cRNA antisense copies were synthesized using T7 polymerase [3].

3. Discussion
DDAH1 was first reported as an enzyme that metabolizes
NG-monomethyl-L-arginine and asymmetrical dimethylarginine to citrulline and methylamine [7] and is known toregulate nitric oxide synthase [5]. A second DDAH isoform(DDAH2) with the same function has since been isolated[4]. DDAH1 expression patterns have been detected in thecentral nervous system (CNS) of adult rats [6], but nodevelopmental profile has been reported histochemically.
Tran et al. [8] has reported that human DDAH1 expression varied little between fetal and adult tissues compared to
DDAH2, but organs which expressed strong DDAH1mRNA signals in our histological results match those in
their dot data results, and some difference between species
is to be expected. Our histological results showed that
DDAH1 mRNA signal expressions in chick and rat embryos
changed radically at each stage, so it may be that not only
DDAH2 but DDAH1 also plays an important role indevelopment.
Although Leiper et al. [4] reported that the expression
pattern of DDAH1 mRNA correlated with nNOS mRNA in
human adult brains, our histological results indicated that, inthe brains of rat embryos, the expression pattern of DDAH1mRNA was different from that of nNOS mRNA (Fig. 9C).
This may be due to the lower correlation between the
DDAH1 mRNA level and DDAH1 protein level, as wasreported by Arrigoni et al. [1]. Further studies will beneeded to address these questions.
References[1] F.I. Arrigoni, P. Vallance, S.G. Haworth, J.M. Leiper, Metabolism ofasymmetric dimethylarginines is regulated in the lung developmentallyand with pulmonary hypertension induced by hypobaric hypoxia, Circulation 107 (2003) 1195 – 1201.
[2] T. Hamada, K. Ui-Tei, Y. Miyata, A novel gene derived from developing spinal cords, SCDGF, is a unique member of the PDGF/VEGF
family, FEBS Lett. 475 (2000) 97 – 102.
[3] S. Harada, T. Imaki, N. Chikada, M. Naruse, H. Demura, Distinct
distribution and time-course changes in neuronal nitric oxide synthaseand inducible NOS in the paraventricular nucleus following lipopolysaccharide injection, Brain Res. 821 (1999) 322 – 332.
[4] J.M. Leiper, J. Santa Maria, A. Chubb, R.J. MacAllister, I.G. Charles,
Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial
arginine deiminases, Biochem. J. 343 (1999) 209 – 214.
[5] R.J. MacAllister, H. Parry, M. Kimoto, T. Ogawa, R.J. Russel, H.
Hodson, G.S. Whitley, P. Vallence, Regulation of nitric oxide synthesis
by dimethylarginine dimethylaminohydrolase, Br. J. Pharmacol. 119(1996) 1533 – 1540.
[6] S. Nakagomi, S. Kiryu-Seo, M. Kimoto, P.C. Emson, H. Kiyama,
Dimethylarginine dimethylaminohydrolase (DDAH) as a nerve-injury-associated molecule: mRNA localization in the rat brain and its
coincident up-regulation with neuronal NO synthase (nNOS) in axotomized motoneuron, Eur. J. Neurosci. 11 (1999) 2160 – 2166.
[7] T. Ogawa, M. Kimoto, K. Sasaoka, Purification and properties of a new
enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat
kidney, J. Biol. Chem. 264 (1989) 10205 – 10209.
[8] C.T.L. Tran, M.F. Fox, P. Vallance, J.M. Leiper, Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison
with DDAH2 and implications for evolutionary origins, Genomics 68(2000) 101 – 105.
[9] K. Yoshida, K. Kawamura, J. Imaki, Differential expression of c-fos
mRNA in rat retinal cells: regulation by light/dark cycle, Neuron 10(1993) 1049 – 1054.