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First published online 26 October 2005
doi: 10.1242/dev.02098

Development 132, 5137-5145 (2005)
Published by The Company of Biologists 2005
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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,{ddagger}

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



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  		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.

 


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  		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.

 


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  		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).

 


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  		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).

 


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  		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).

 


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  		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).

 


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  		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).

 




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