Anatomia Histologia Embryologia

ORIGINAL ARTICLE

Gene Expression of Spag6 in Chick Central Nervous System
T. Hamada1,2*, M. Teraoka3, J. Imaki4, K. Ui-Tei5, R. K. Ladher3 and T. Asahara2
Addresses of authors: 1 Department of Anatomy and Neurobiology, National Defense Medical Collage, 3-2 Namiki, Tokorozawa, Saitama359-8513;
2Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, 2-2 Minatojima Minami-machi, Chuo-ku,
Kobe, Hyogo 650-0047;
3
Sensory Development Laboratory, RIKEN Kobe Institute, Center for Developmental Biology, 2-2-3 Minatojima Minami-machi, Chuo-ku,
Kobe, Hyogo 650-0047;
4
Department of Developmental Anatomy and Regenerative Biology, National Defense Medical College, 3-2, Namiki, Tokorozawa,
Saitama 359-8513;
5
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo
113-0033, Japan*Correspondence:
Tel.: +81 4 2995 1478;
fax: +81 4 2996 5186;
e-mail: thamada@ndmc.ac.jp

With 5 figures
Received December 2009; accepted for
publication March 2010
doi: 10.1111/j.1439-0264.2010.01000.x

Summary
Using a differential display method, we identified sperm-associated antigen 6(Spag6) as a gene with a dynamic expression profile within the chick embryonic spinal cord. The expression of Spag6 gradually decreased along with spinal
cord development. Spag6 expression was detected adjacent to the ventricularzone of the spinal cord at embryonic day (E) 4. At E6, Spag6 was apparent in
the ventral ventricular zone adjacent to floor plate and the surrounding region
close to the ventricular zone, with additional weak expression at the adjacentregion to the ventral horn. At E10, the Spag6 mRNA can be detected slightly inthe ventral ventricular zone and surrounding region of dorsal ventricular zone.
In the E6 hindbrain, Spag6 was detected in the roof, the ventricular zone adjacent to floor plate and the surrounding regions of the ventricular zones. In theE6 caudal diencephalon, Spag6 expression was detected adjacent to the ventricular zone. As Spag6 was expressed in areas containing ependymal progenitor
cells and in the borders of undifferentiated regions, Spag6 may be involved in
the development of ependymal cells and in the differentiation process of neuronal cells in chick neural organs.

Introduction
To understand the functions of the nervous system inmaturity, it is important to determine the mechanisms bywhich it is formed. During neural development, cells differentiate to specific types of neurons and undergo migration, synapse formation and cell death precisely, withrespect to time and location (Edelman, 1983; Reichardt,
1984; Goodman and Shatz, 1993; Tanabe and Jessell,1996). Accumulating evidence indicates that morphological differentiation and cell motility are based on manykinds of molecules, which are located on cell surfaces,
secreted from cells and within the cells, such as cytoskeletal molecules (Edelman, 1983; Riederer, 1990; Tanabe andJessell, 1996). Therefore, one of the approaches to revealª 2010 Blackwell Verlag GmbH • Anat. Histol. Embryol. 39 (2010) 227–232

the mechanisms underlying neural differentiation is to
identify molecules, the expression of which changes during development.
The development of the spinal cord has been extensively studied morphologically using chick embryos(Hamburger, 1948). It is well known that developmentally
important phenomena occur in the spinal cord from E4
to E10, examples of which include natural occurring cell
death of motoneurons, and glial cell differentiation andmigration (Hamburger, 1975; Miller et al., 1997).
The present study was conducted to isolate molecules
related to differentiation of the spinal cord in chickembryos. To realize this aim, using the differential display
procedure, we isolated several genes, expressions of whichchanged drastically from E4 to E10. Among them, in this227

Spag6 in Chick CNS

article, we report the expression pattern of sperm-associated antigen 6 (Spag6) (Neilson et al., 1999; Sapiro et al.,2000) in chick spinal cord. Our data indicated that theexpression levels of Spag6 decreased according to spinal
cord development. Furthermore, in situ hybridization
showed that Spag6 was expressed in areas containing
ependymal progenitor cells and in the borders of undifferentiated neural regions.

T. Hamada et al.

were obtained and their full-length open reading frames
were identified using 5¢-rapid amplification of the cDNA
ends using the 5¢-RACE System, version 2.0 (Invitrogen,
Carlsband, USA) and cDNA library screening (Hamada
et al., 2000).
Reverse transcription (RT)-PCR

Animals
Fertilized white leghorn chicken eggs were purchased froma local poultry (Miyake Eggs, Shizuoka, Japan). The eggs
were incubated at 37C in a humidified egg incubator.

RT-PCR was performed according to the method describedin Sambrook and Russell, 2001. The paired primers used(sense/antisense) were as follows: for Spag6, GAAGACATA
CTCCTGAGCAT/ACACAGCTTGCTGGAAATAC [583-basepairs (bp) PCR product]; for Glyceraldehyde phosphate
dehydrogenase (GAPDH), GGCTGCTAAGGCTGTGGGGA/TATCAGCCTCTCCCACCTCC [546-bp PCR product].

RNA isolation, differential display and cDNA cloning

Northern blotting analysis

Preparation of total RNA from chick organs, for differential
display, molecular cloning and Northern blotting analysis,
was performed by LiCl precipitation method. The differential display method was performed using the RNAimage
kit (GenHunter, Nashville, TN, USA) with Ampli Taq Gold
(Perkin-Elmer, Waltham, MA, USA) and [a-35S]dATP(NEN). Several differentially expressed cDNA fragments

Total RNAs (20 lg) were run on a denaturing formaldehyde-agarose gel and transferred to nylon membrane.
RNAs blotted onto the membrane was hybridized with a[32P]-labelled Spag6 cDNA at 65C in a buffer containing
0.5 m sodium phosphate (pH 7.2), 7% SDS and 1 mm
EDTA. The membrane was washed twice by immersing ina 0.2 · SSC and 0.1% SDS solution for 40 min at 65C.

Methods

Fig. 1. Deduced amino acid alignments of chick, human and mouse Spag6 (Neilson et al., 1999; Sapiro et al., 2000). Identical amino acids are
boxed. Numbers on the right indicate amino acid positions relative to the initiator methionine residue.

228ª 2010 Blackwell Verlag GmbH • Anat. Histol. Embryol. 39 (2010) 227–232

T. Hamada et al.

Spag6 in Chick CNS

The membrane was analysed using a BAS2000 system(Fuji, Tokyo, Japan) and exposed to an X-ray film.
In situ hybridization

Fig. 2. Reverse transcription-PCR analysis of the change in chickSpag6 expression according to spinal cord development. Developmental stages are indicated in each lane. GAPDH was used as control.

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ve
Li

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Lu

in
Sk

Ey
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ai

Br

in

al

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rd

(Fig. 2). Weak Spag6 expression could be detected until
postnatal day (P) 1 and was not apparent thereafter.
Next, we examined the expression of Spag6 mRNA at
E10 in neural organs, i.e. the spinal cord and brain, andin several other organs by Northern blotting analysis
using chick Spag6 cDNA as a probe. Northern blot analysis against total RNAs from spinal cord and brain of E10

Sp

Chick embryos were fixed in paraformaldehyde. Frozen
specimens were sectioned with a cryostat at 20 lm thickness and mounted onto silanize-coated slides. In situ
hybridization was performed with RI- single strand Spag6
RNA according to a previous report (Yoshida et al.,1993). Briefly, sections were dried for 0.5–1 h andimmersed in 1% Triton X-100 in 50 mm Tris/HCl buffer(pH 8.0) containing 25 mm EDTA for 30 min and acetylated with 0.25% acetic anhydride in 0.1 m triethanolamine. The sections were dehydrated in an ascending
ethanol series and air-dried. The probe (1 · 106 cpm/ml)
was dissolved in a buffer containing 50% formamide,
10% dextran, 1· Denhart’s solution (0.02% each of Ficoll
400, polyvinylpyrrolidone, and bovine serum albumin),
12 mm EDTA (pH 8.0), 10 mm Tris/HCl (pH 8.0),
30 mm NaCl, 0.5 mg/ml yeast tRNA, and 10 mm dithiothreitol, and 100 ll of probe solution was applied toeach slide. The slides were coverslipped and incubated at
65C overnight. The coverslips were then removed andthe slides were rinsed in 4· SSC (1· SSC is 0.15 m NaCl,
0.015 m Na-citrate, pH7.5), digested with RNAse A(20 mg/ml) for 30 min at 37C, and rinsed sequentiallyin 2· SSC, 1· SSC, 0.5· SSC, then in 0.1· SSC for
30 min at 65C. The sections were exposed to X-ray films(Kodak BioMax, Rochester, NY, USA) for 3–4 days, thendipped in NTB2 nuclear emulsion (1:1 with water;
Kodak), and then exposed for 1–3 weeks. Counter staining was performed with thionin.
Results

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One approach to understand the mechanisms underlying
neural development is to search for a molecule(s), theexpression of which dramatically changes over certain
developmental windows. To identify such molecules, weemployed a differential display method using the chickspinal cord from embryonic day (E) 4 to E10. One of thegenes the expression of which gradually decreased fromE4 to E10 was chick sperm-associated antigen 6 (Spag6)(GenBank accession no. XM418607). The chick Spag6 hasan open reading frame of 1527 nucleotides encoding aputative protein composed of 508 amino acids (Fig. 1).
The amino acid sequence of chick Spag6 showed 87.4 and85.8% homology to mouse and human Spag6 (Neilson
et al., 1999; Sapiro et al., 2000) respectively (Fig. 1).
We analysed the developmental change of Spag6 expression in chick spinal cord by RT-PCR. Spag6 expression wasapparent until E6 and decreased gradually subsequently

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28 S

18 S

Fig. 3. Northern blot analysis of Spag6 mRNA expression in various
chick tissues at E10. Spag6 bands are indicated by arrows. The lower
panel shows ethidium bromide-stained 18S and 28S rRNAs as control.

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T. Hamada et al.

chick detected two bands (4.5 and 3.8 kb) of Spag6
mRNAs, indicating that Spag6 may have alternative splicing variants, transcripts with different poly (A) lengths or
two poly (A) additional sites. At E10, Spag6 expression
was also detected in the kidney, gut and heart (data notshown) (Fig. 3).
We next examined the detailed expression pattern of
Spag6 in the chick embryonic central nervous system byin situ hybridization. In the E4 spinal cord, Spag6 mRNAwas detected in the region adjacent to the ventricularzone (Fig. 4a,b). At E6, Spag6 was expressed in the ventral
ventricular zone adjacent to the floor plate and in thearea surrounding the dorsal ventricular zone. Weakexpression in the region adjacent to the ventral horn wasobserved (Fig. 4c,d). At E10, Spag6 mRNA can bedetected in the ventral ventricular zone and surrounding
region of the dorsal ventricular zone (Fig. 4e,f). In the E6
hindbrain, Spag6 signals were also detected in the ventricular zone adjacent to the floor plate and in the surrounding regions of the ventricular zone and roof (Fig. 5a,b).
In the fore- and midbrain, expression of Spag6 was

(a)

(b)

(c)

(d)

(e)

230

(f)

detected clearly in a region adjacent to the ventricularzone of the caudal diencephalon (Fig. 5c,d). The retina
appeared whitish because of the reflection of pigmentation under dark field optics.
Discussion
This is the first report documenting Spag6 expression inembryonic neural organs. Spag6 is found in the testis,specifically in the sperm flagella and cells in spermatogenesis process and has an important role in sperm motility(Neilson et al., 1999; Sapiro et al., 2000). Recently, Spag6
expression was reported in tumour and undifferentiatedproliferating cells in vitro (Shen et al., 2006; Steinbach etal., 2006). However, there are no reports documenting
the expression of Spag6 in embryonic tissues in detail.
In postnatal mouse, Spag6 localizes to the surface of thebrain ventricles, and Spag6 knockout mice phenotypes
have hydrocephalus, arising secondary to defects in motility of the ependymal cell cilia, which line the lumen of theneural tube and form from the ventricular cells (Sapiro

Fig. 4. In situ hybridization
analysis of chick Spag6 mRNAin the embryonic spinal cord at
E4 (a and b), E6 (c and d) andE10 (e and f). (a, c and e) aredark field and (b, d and f) arebright field photomicrographs.
(a) and (b, c and d), and(e and f) were taken from thesame slice. fp, floor plate; ml,mantle layer; vh, ventral horn;vz, ventricular zone. Scalebar = 100 lm.

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T. Hamada et al.

Fig. 5. In situ hybridization
analysis of chick Spag6 mRNAin the embryonic brain at E6.
(a) and (c) are dark field and(b) and (d) are bright field
photomicrographs. (a) and (b)(hindbrain), (c) and (d) (foreand midbrain) were taken fromthe same slice. fp, floor plate;vz, ventricular zone; rf, roof;
rt, retina; t, telencephalon;d, diencephalon; m, mesencephalon. Scale bar = 250 lm.

Spag6 in Chick CNS

(a)

(b)

(c)

(d)

et al., 2002; Zhang et al., 2007). In addition, our results
showed that Spag6 was expressed in the roof of the hindbrain and in the spinal ventral ventricular zone adjacentto the floor plate at E6 (Figs 4 and 5), which contains
ependymal progenitor cells (Fu et al., 2003). Our datasuggest that Spag6 is expressed from the early stages of
ependymal cell development and may play a role in axoneme formation of ependymal cell cilia, such as in sperm
flagella formation during spermatogenesis. However, weobserved that Spag6 was not only expressed in ependymal
progenitor cells but also in areas other than the ventricular
cells of embryonic spinal cord and brain. Furthermore, it
was reported that Spag6 is expressed in an artificial in vitro
culture of undifferentiated human neural stem cells, theexpression of which decreases according to the number ofcell passages (Shen et al., 2006). This cited report corroborates our result. Moreover, at E4, Spag6 was expressed in
the surrounding region of the ventricular zone, but not inthe ventricular zone itself or in the lateral mantle layer(Fig. 4). In the embryonic spinal cord, the ventricular zone
contains undifferentiated neural cells, whereas the lateralmantle layer has differentiated cells. Our data suggest thatSpag6 might act in the differentiation process of neuralcells in chick embryonic spinal cord as Spag6 is mainly
expressed in the intermediate region of the abovementioned areas, and expression decreased according toprogression of spinal cord development. Furthermore,as the role of microtubules in neural development anddifferentiation is well known (Riederer, 1990) and theinteraction between Spag6 and microtubules has beenpreviously documented (Sapiro et al., 2000), Spag6
might play roles associated with microtubules for neuraldifferentiation.
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Acknowledgement
We thank Dr S. Minami and T. Imaki for the use of theirequipments.
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