First published online 26 October 2005
doi: 10.1242/dev.02098
Development 132, 5137-5145 (2005)
Published by The Company of Biologists 2005
Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation
Cha-Gyun Jung1,*,
Hye-Jung Kim1,*,
Makoto Kawaguchi2,
Kum Kum Khanna3,
Hideki Hida1,
Kiyofumi Asai4,
Hitoo Nishino1 and
Yutaka Miura4,
1 Department of Neurophysiology and Brain Science, Graduate School of Medical
Sciences, Nagoya City University, Mizuhoku, Nagoya 467-8601, Japan
2 Department of Pathology, Niigata Rosai Hospital, Japan Labor Health and
Welfare Organization, 1-7-12 Tooun-cho, Jhoetsu, Niigata 942-8502, Japan
3 Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane
4029 Queensland, Australia
4 Department of Molecular Neurobiology, Graduate School of Medical Sciences,
Nagoya City University, Mizuhoku, Nagoya 467-8601, Japan
View larger version (67K):
[in a new window]
|
Fig. 1. ATBF1 expression is associated with neuronal differentiation in E14.5 rats.
(A) BrdU (green) and ATBF1 (red). ATBF1 is expressed in the ganglionic
eminence (GE), medial tegmentum (Tg), isthmus (I), pons (P), colliculus (C),
spinal cord (S), tongue (T) and elsewhere. (B) mRNA expression in embryonic
and postnatal rat brains evaluated by real-time RT-PCR. (C1-F2) Magnification
of the GE and further magnification of the framed area. (C1) BrdU labeling in
the VZ and SVZ. ATBF1 is present in the differentiating field (DF) but not in
the white matter (W). (C2) Higher magnification of the boxed area in C1. (D1)
Nestin is present in the VZ and SVZ extending a fibrous network from the SVZ
into the DF, but is segregated from ATBF1-positive cells. (D2) Higher
magnification of the boxed area in D1. (E1) ß-tubulin III is present in
the DF with ATBF1. (E2) Higher magnification of the boxed area in E1. (F1)
MAP2 is present in the DF with ATBF1. (F2) Higher magnification of the boxed
area in F1.
|
|
View larger version (77K):
[in a new window]
|
Fig. 2. Overexpression study of ATBF1 cDNA in primary cultured neuroepithelial
cells. (A1-5) Immunohistochemistry for nestin (green) overlaps with the HA-tag
(red) after transfection of an HA-tagged mock expression vector. (B1-5) Nestin
(green) expression is suppressed by HA-tagged ATBF1 (red) expression. (C1-5)
ß-Tubulin (green) does not overlap with an HA-tagged (red) mock
expression vector. (D1-5) ß-tubulin (green) expression overlaps with
HA-tagged ATBF1 (red) expression. (E) A nestin-specific enhancer element is
suppressed by ATBF1. (F) The Neurod1 promoter is activated by ATBF1.
*P<0.01.
|
|
View larger version (65K):
[in a new window]
|
Fig. 3. ATBF1 induces cell cycle arrest in Neuro 2A cells without inducing
apoptosis. (A1-5) HA-tag (red) and BrdU (green) overlap (yellow). (B1-5) BrdU
(green) does not overlap with HA-tagged ATBF1 (red) expressions. The HA-tagged
ATBF1 positive cells appear with neurite like projections. (C1-5) TUNEL
staining (green) after transfection with an HA-tag (red) mock expression
vector. (D1-5) TUNEL-positive cell (green) is small compared with HA-tagged
ATBF1-positive cells that appear with larger cell bodies and neurite-like
projections. (E1-4) FACS analysis after transfection with HA-tag or HA-tagged
ATBF1 expression vector. The cells expressing HA-tag or HA-tagged ATBF1 were
selected by gating the higher intensity of the green signal (gate: G1
indicated in E1 and E2). PI patterns of selected cells expressing HA-tag (E3)
and HA-tagged ATBF1 (E4).
|
|
View larger version (68K):
[in a new window]
|
Fig. 4. PI patterns of P19 cells by FACS analysis corresponding to undifferentiated
P19 cells (A), P19 cells after RA treatment for 24 hours in floating culture
(B) and differentiated P19 cells on an adhesive dish after 7 days (C).
Immunoreactivities of P19 cells for ATBF1 (red) and ß-tubulin (green),
overlaid with DAPI staining (blue). Undifferentiated P19 cells (A1). P19 cells
after RA treatment for 24 hours in floating culture (B1). Differentiated P19
cells on an adhesive dish after 7 days (C1). ATBF1 (red) and ß-tubulin
(green) observed by confocal microscopy. P19 cells express ATBF1 in the
cytoplasm after RA treatment for 24 hours in floating culture (B2-4).
Differentiated P19 cells express ATBF1 in the nucleus on an adhesive dish
after 7 days treatment with RA (C2-4).
|
|
View larger version (102K):
[in a new window]
|
Fig. 5. Immunohistochemistry for ATBF1 (red, A,B) counterstained with the general
membrane dye DiOC6(3) (green, A,B) in RA-treated P19 cells. (A)
Subcellular localization of ATBF1 in the adhesive condition. Nuclear
localization of ATBF1 on dishes coated with poly-L-onetime (A1),
poly-L-ornithine+fibronectin (A2), poly-L-ornithine+laminin (A3),
poly-L-lysine (A4) and gelatin (A5) at 3 hours after cell transfer to the
dishes. Cells pre-treated with LY294002 (20 µM, A6), ryanodine (100 nM, A7)
and caffeine (5 mM, A8). (B) Subcellular localization of ATBF1 in floating
conditions. ATBF1 is localized in the cytoplasm of cells in the aggregated and
floating conditions (B1). Treatment with leptomycin B (10 nM, 1 hour, B2)
induces ATBF1 accumulation in the nucleus in the floating condition. ATBF1 is
localized in the nucleus of cells separated from embryonic bodies after
treatment with EDTA (1 mM, 30 minutes) and pipette (B3).
|
|
View larger version (77K):
[in a new window]
|
Fig. 6. ATBF1 expression in the midline region of the E14.5 rat brain. ATBF1 is
expressed in the neuroepithelial cells at the curved regions and the narrowing
part of the brain. (A) Plates showing sagittal (B,C) and horizontal (D)
sections of the brain. (B1,C1,D1) Higher magnification of the boxed areas in
B,C,D, respectively. ATBF1 is expressed at high levels in tegmantum (B1),
thalamus (C1) and center of the isthmus (D1).
|
|
View larger version (93K):
[in a new window]
|
Fig. 7. ATBF1 expression at the GE and the isthmus in a sagittal section of E14.5
rat brain. (A) ATBF1 is expressed at the GE; in the cytoplasm of the VZ;
weakly in the nucleus of the SVZ; strongly in the nucleus of the DF; absent
from the white matter (W). (A1-A3) Higher magnification of the numbered areas
in A. (B) Fibronectin is expressed on the surface of the ventricle, sparsely
in the SVZ, in intercellular space of the DF and strongly in the white matter
(W). (B1-B3) Higher magnification of the numbered areas in B. (C) Expression
pattern of fibronectin at the isthmus. (C1) Higher magnification of the framed
area of C. There are two regions: one has a high cell density (C1, region I),
the other has a lower cell density (C1, region II). (A,B,C,C1) DAB and
Hematoxylin counterstaining. BrdU (D,D1,D2), ATBF1 (E,E1,E2) and merged images
of BrdU and ATBF1 (F,F1,F2). BrdU labeling indicates rapid proliferation on
the ventricular side (D1) and suppression of the cell cycle on the dorsal side
(D2). ATBF1 expression is present in the cytoplasm (E1) and the nucleus
(E2).
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2005
