Mechanisms of Development 118 (2002) 209–213
www.elsevier.com/locate/modo
Gene expression pattern
NZF-2b is a novel predominant form of mouse NZF-2/MyT1, expressed in
differentiated neurons especially at higher levels in newly generated ones
Fumio Matsushita*, Toshiki Kameyama, Tohru Marunouchi
Division of Cell Biology, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan
Received 8 April 2002; received in revised form 31 May 2002; accepted 11 July 2002
Abstract
NZF-2 (MyT1) is a member of C2HC-type zinc finger transcription factors. A novel form of mouse NZF-2 has been isolated. This novel
form, NZF-2b, has an additional C2HC-type zinc finger motif. The expression levels of NZF-2b are by far the more predominant than those
of the already known form of NZF-2. In embryonic mouse nervous system, the expression of NZF-2b starts as early as at 9.5 days post-coitum
(dpc) in newly differentiated neurons in the central nervous system (CNS) and the peripheral nervous system (PNS). q 2002 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Mouse; NZF-2; MyT1; C2HC-type zinc finger; Transcription factor; Development; Neuronal differentiation; Neurogenesis; Spinal cord; Dorsal
root ganglia; Brain; Central nervous system; Peripheral nervous system; Peripheral ganglia
1. Results and discussion
Neural zinc finger proteins (NZFs) are transcription
factors with DNA-binding domains of a C2HC-type zinc
finger motif (Jiang et al., 1996; Kim and Hudson, 1992;
Weiner and Chun, 1997; Yee and Yu, 1998). Xenopus
NZF-2 (X-MyT1) has a regulatory function in neuronal
differentiation (Bellefroid et al., 1996). In mammals, the
function of NZF-2 has been suggested mainly in relation
with gliogenesis (Armstrong et al., 1995; Kim and Hudson,
1992). Despite some indications that NZF-2 is also functional in neuronal differentiation in rat, the previous histological analysis of rat was limited to advanced stages of
central nervous system (CNS) development (Kim et al.,
1997). Therefore, we focused on the expression of NZF-2
in early stages of neuronal differentiation in mammals. This
work shows that NZF-2b, a novel predominant subtype of
mouse NZF-2 with an additional zinc finger, is expressed in
differentiated neurons especially at higher levels in newly
generated ones.
1.1. Isolation of cDNA encoding NZF-2b
By using partial cDNA fragment of the already known
form of mouse NZF-2/MyT1 (nucleotides (nt) 628–1155;
Kim et al., 1997) as a probe, we isolated cDNAs encoding
a novel subtype of mouse NZF-2 from embryonic mouse
brain cDNA library (ICR, 17.5 dpc; Kameyama et al., 1996;
Fig. 1A). We designate this novel subtype as NZF-2b
(DDBJ/GenBank accession number AB082378) and the
already known form as NZF-2a. NZF-2b has an additional
C2HC-type zinc finger motif at N-terminus, but otherwise is
almost identical with NZF-2a (Fig. 1B, C). In the databases,
cDNA homologous to NZF-2b is found only in human
(KIAA0835 gene; Nagase et al., 1998; Fig. 1B, C), and
cDNA homologous to NZF-2a is found in rat and Xenopus.
1.2. NZF-2b is a predominant form of mouse NZF-2
To investigate expressional differences of two types of
NZF-2, we estimated the expression levels of both types of
NZF-2 transcripts in the course of development by using
quantitative real time reverse transcriptase-polymerase
chain reaction (RT-PCR). The result reveals that the copy
numbers of NZF-2b are by far the more predominant than
those of NZF-2a in mRNA level at all stages of development
and in adulthood (Fig. 2). NZF-2a is expressed at extremely
low levels. False detection of NZF-2a due to contamination
of genomic DNA in the RT-PCR reaction is excluded
because NZF-2a is not detected without reverse transcriptase.
1.3. Expression of NZF-2b in the spinal cord
* Corresponding author. Tel.: 181-562-93-9378; fax: 181-562-93-8834.
E-mail address: [email protected] (F. Matsushita).
We focused on the early stages of neuronal differentiation
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0925-477 3(02)00250-2
210
F. Matsushita et al. / Mechanisms of Development 118 (2002) 209–213
Fig. 1. (A) Schematic representations of mouse NZF-2 cDNAs. Previously reported mouse NZF-2a/MyT1 cDNA (Kim et al., 1997), the probe that we used to
screen, and the resultant isolated clones are shown. Respective open reading frames (ORFs) of NZF-2a and NZF-2b (clone 7N-3) are indicated by boxed
arrows. The regions specific to NZF-2a and NZF-2b are shown in green and red, respectively. Note that all five isolated clones encode NZF-2b. (B) Structural
comparison of mouse NZF-2 proteins. The black boxes and the hatched boxes indicate C2HC-type zinc finger units and the acidic domains, respectively. The
specific regions are shown in the same way as in (A). Percentages of identity between NZF-2b and each of two other proteins are shown for their corresponding
domains (set apart by straight lines). The entire NZF-2b protein shows 87% identity with NZF-2a (Kim et al., 1997) and 90% identity with the deduced product
of human KIAA0835 cDNA (Nagase et al., 1998). (C) Amino acid sequence alignment of N-terminal portion of mouse NZF-2a, NZF-2b, and the deduced
protein of human KIAA0835 cDNA clone. Identical residues are shown by shaded boxes. The conserved cysteine and histidine residues that constitute the
C2HC-type zinc finger are indicated by asterisks.
and analyzed the expression pattern of NZF-2b from 9.5 to
13.5 dpc mouse nervous system. NZF-2b transcripts are
detected in the earliest born neurons. At 9.5 dpc, they are
detected in the ventrolateral part of the spinal cord, which
later become motor neurons (arrowhead in Fig. 3C; Altman
and Bayer, 1984; Garel et al., 1997). They are also detected
in the dispersed cells of the alar plate interneurons (arrows
in Fig. 3C). During spinal cord development, the expression
level of NZF-2b is highest in the latest born neurons (the
subventricular zone (SVZ); arrowheads in Fig. 3G, K). In all
stages analyzed, the signals of a probe specific to NZF-2b
are almost identical with those of a probe common to NZF2a and NZF-2b (NZF-2com, compare Fig. 3B with C, etc).
No detectable signals are observed by using a probe specific
to NZF-2a (Fig. 3D, H, L).
2com probe to show further the expression of NZF-2b in the
developing nervous system. NZF-2b transcripts are detected
in the early differentiated neurons within the neuroepithelium and the neural crest cells at 9.5 dpc (data not shown).
At 12.5 dpc, the transcripts of NZF-2b are detected in the
differentiated neurons within the forebrain (Fig. 4A),
midbrain, and hindbrain (Fig. 4B). In these neurons, the
expression level of NZF-2b is highest in the latest born
neurons (SVZ, arrows in Fig. 4A, B). NZF-2b is also
detected in the differentiated neurons of the sensory organs
(Fig. 4C, D) and the peripheral ganglia (Fig. 4E, F).
2. Methods
2.1. Real time RT-PCR analysis
1.4. Expression of NZF-2b in the other CNS and the PNS
Not only in the spinal cord, but also in the other CNS and
in the PNS, the signals of NZF-2com probe are almost identical with those of NZF-2b. In consequence, we used NZF-
Quantitative real time RT-PCR analysis was performed
by LightCycler (Roche Diagnostics) with QuantiTect SYBR
Green PCR kit (Quiagen). Total RNAs from various sources
of ddy mice were used as templates for Omniscript reverse
F. Matsushita et al. / Mechanisms of Development 118 (2002) 209–213
211
Fig. 2. Quantitative real time RT-PCR analysis of the transcripts of NZF-2 subtypes. Copy numbers of transcripts are shown by the bars for sets of primers specific
to NZF-2a (black bars), specific to NZF-2b (hatched bars), and common to NZF-2a and NZF-2b (NZF-2com, open bars). At all stages quantified, the copy
numbers of NZF-2b transcripts are almost equal to those of NZF-2com transcripts and are more than 65-fold excess of those of NZF-2a transcripts. The expression
levels of NZF-2b have a peak at 15.5 dpc and keep low in adulthood. Transcripts were quantified by comparison with simultaneous standard reactions against
linearized plasmids containing cDNAs of respective targets. The final copy number of transcripts shown in vertical axis was normalized with 2.0 £ 10 5 molecules
of transcripts of the house keeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH), which correspond to the copy number in about 10 ng total
RNAs. The sources of total RNAs were heads at 10.5 dpc, whole brains at 12.5, 15.5, and 18.5 dpc, and also whole brains at postnatal day 2 (P2), P7, P14, P21 and
adult mice. The error bars indicate the standard error.
Fig. 3. The expression of NZF-2b within developing spinal cord by RNA in situ hybridization. Transverse sections of 9.5 dpc (A–D), 11.5 dpc (E–H), and 13.5 dpc
(I–L) spinal cord hybridized with indicated probes or immunostained with TuJ1 (Sigma), a monoclonal antibody against neuron-specific class III b-tubulin, which
detects differentiated neurons (Easter et al., 1993) are shown. At 9.5 dpc, NZF-2b transcripts are detected in the earliest born neurons (an arrowhead and arrows in C).
At 11.5 dpc, NZF-2b is expressed at high levels in the SVZ (arrowheads in G), at low levels in the ML, and at very low levels in the differentiated MN. At 13.5 dpc,
NZF-2bis expressedin the SVZ andthe ML, at higherlevelsin the SVZ ofthe alarplate(arrowheadsin K). NomRNAis detectedin the whitematter.DRG, dorsal root
ganglia; ML, mantle layer; MN, motor neurons; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bar, 0.1 mm (A–L).
212
F. Matsushita et al. / Mechanisms of Development 118 (2002) 209–213
Fig. 4. In situ hybridization with NZF-2com probe in 12.5 dpc mouse embryo. Coronal sections of forebrain (A), midbrain and hindbrain (B), eye (C), nasal
cavity (D), and peripheral ganglia (E,F) are shown. In all figures, dorsal is to the top. The expression level of NZf-2b is especially high in the subventricular
zone (arrows in A and B). AP, anterior pons; BG, basal ganglia; CBL, cerebellar primordium; CTX, cortex; Med, medulla; HT, hypothalamus; IS, isthmus; OE,
olfactory epithelium; RE, respiratory epithelium; RET, retina; SC, superior colliculus; SCG, superior cervical sympathetic ganglion; ST, sympathetic trunk;
TG, tegmentum; TH, thalamus; VNO, vomeronasal organ; Vg, trigeminal ganglion; VIIg, facial ganglion; VIIIg, vestibulocochlear ganglion; IXg, inferior
ganglion of glossopharyngeal (IX) nerve; Xg–XIg, superior ganglion of vagus (X)-cranial accessory (XI) ganglion complex. Scale bars: 0.5 mm (A,B); 0.1 mm
(C–F).
transcriptase (Quiagen). Sets of primers for NZF-2com (5 0 ATACCTCTGTCCAGAAGGCG-3 0 ,
5 0 -TGTCATCAT0
0
CAGAGCGAACC-3 ), NZF-2a (5 -AATCCAGGCAGAAGGCTGA-3 0 ,
5 0 -TGTTTCCAGGTCTCTGCTCC0
0
3 ), NZF-2b (5 -GCAGTGAGGATGCTGAAGTG-3 0 , 5 0 GGGTGATGAATCTCCTCCTGT-3 0 ), and GAPDH (5 0 ATTGTGGAAGGGCTCATGAC-3 0 , 5 0 -ATGCAGGGATGATGTTCTGG-3 0 ) give specific RT-PCR products of
102, 128, 106, and 121 bp, respectively.
nt 5–774 of NZF-2a cDNA and to nt 1–788 of NZF-2b
cDNA.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research from JSPS and one from FHU, and Grants-in-Aid
for FHU High-tech Research Center and for FHU Bioventure Research Center each from MEXT and FHU.
2.2. In situ hybridization and immunohistochemistry
References
Serial paraffin sections (10 mm) were processed. NZF2com probe was a mixed probe made from two fragments
of NZF-2b cDNA: one, nt 1947–2801, which separates the
two zinc finger clusters, and the other corresponding to nt
3790–5510 of the 3 0 untranslated region. The two subtypespecific probes were made from fragments corresponding to
Altman, J., Bayer, S.A., 1984. The development of the rat spinal cord. Adv.
Anat. Embryol. Cell. Biol. 85, 1–164.
Armstrong, R.C., Kim, J.G., Hudson, L.D., 1995. Expression of myelin
transcription factor I (MyTI), a ‘zinc-finger’ DNA-binding protein, in
developing oligodendrocytes. Glia 14, 303–321.
Bellefroid, E.J., Bourguignon, C., Hollemann, T., Ma, Q., Anderson, D.J.,
Kintner, C., Pieler, T., 1996. X-MyT1, a Xenopus C2HC-type zinc
F. Matsushita et al. / Mechanisms of Development 118 (2002) 209–213
finger protein with a regulatory function in neuronal differentiation.
Cell 87, 1191–1202.
Garel, S., Marin, F., Mattei, M.G., Vesque, C., Vincent, A., Charnay, P.,
1997. Family of Ebf/Olf-1-related genes potentially involved in neuronal differentiation and regional specification in the central nervous
system. Dev. Dyn. 210, 191–205.
Jiang, Y., Yu, V.C., Buchholz, F., O’Connell, S., Rhodes, S.J., Candeloro,
C., Xia, Y.R., Lusis, A.J., Rosenfeld, M.G., 1996. A novel family of
Cys–Cys, His–Cys zinc finger transcription factors expressed in developing nervous system and pituitary gland. J. Biol. Chem. 271, 10723–
10730.
Kameyama, T., Murakami, Y., Suto, F., Kawakami, A., Takagi, S., Hirata,
T., Fujisawa, H., 1996. Identification of a neuronal cell surface molecule, plexin, in mice. Biochem. Biophys. Res. Commun. 226, 524–529.
Kim, J.G., Armstrong, R.C., Agoston, D.v., Robinsky, A., Wiese, C., Nagle,
J., Hudson, L.D., 1997. Myelin transcription factor 1 (Myt1) of the
oligodendrocyte lineage, along with a closely related CCHC zinc finger,
213
is expressed in developing neurons in the mammalian central nervous
system. J. Neurosci. Res. 50, 272–290.
Kim, J.G., Hudson, L.D., 1992. Novel member of the zinc finger superfamily: a C2–HC finger that recognizes a glia-specific gene. Mol. Cell.
Biol. 12, 5632–5639.
Nagase, T., Ishikawa, K., Suyama, M., Kikuno, R., Hirosawa, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., Ohara, O., 1998. Prediction of the coding sequences of unidentified human genes. DNA Res. 5,
355–364.
Weiner, J.A., Chun, J., 1997. Png-1, a nervous system-specific zinc finger
gene, identifies regions containing postmitotic neurons during mammalian embryonic development. J. Comp. Neurol. 381, 130–142.
Yee, K.S., Yu, V.C., 1998. Isolation and characterization of a novel
member of the neural zinc finger factor/myelin transcription factor
family with transcriptional repression activity. J. Biol. Chem. 273,
5366–5374.