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First published online 3 September 2003
doi: 10.1242/dev.00724

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CBF1 controls the retinotectal topographical map along the anteroposterior axis through multiple mechanisms

Division of Molecular Neurobiology, National Institute for Basic Biology, and Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki 444-8585, Japan

View larger version (70K): [in a new window]   Fig. 1. Expression of ephrin A5 and ephrin A2 in the developing chick retina. Whole-mount and section in situ hybridization of HH stage 12 (A), HH stage 18 (B), E4 (D) and E8 (C,E) chick embryos with digoxigenin-labeled riboprobes. Samples were hybridized with antisense probes for ephrin A5 (AC) or ephrin A2 (D,E). (C, parts b,c; E, parts b,c) are enlargements of the nasal and temporal areas boxed in the left panels (C, part a; E part a), respectively. In section in situ hybridization, nasal (N, anterior) is upwards, and temporal (T, posterior) is downwards. Scale bars: 100 µm in B, part b; 600 µm in C, part a and E, part a; 200 µm in D.

View larger version (77K): [in a new window]   Fig. 2. Misexpressed CBF1 induces expression of SOHo1 and GH6, and represses expression of EphA3 and CBF2. Whole-mount in situ hybridization of E3 chick embryos (HH stage 18 to 20) transfected with CBF1/RCAS. Embryos were hybridized with antisense probes for SOHo1 (A,B), GH6 (C,D), EphA3 (E,F) or CBF2 (G,H). The untransfected sides (control) are shown in A,C,E,G and the contralateral transfected sides of the same embryo are shown in B,D,F,H as inverted images for ease of comparison. Arrowheads indicate the border of the endogenous expression area on the control side.

View larger version (72K): [in a new window]   Fig. 3. Misexpressed CBF1 induces expression of ephrin A5 and ephrin A2, and repressed that of EphA3 and CBF2. (A,B,F,G,K,L,P,Q) Horizontal section in situ hybridization of control retinae (A,F,K,P) and CBF1/RCAS-electroporated reginae (B,G,L,Q) of E8 embryos. (D,E,I,J,N,O,S,T) Respective enlargements of the boxed temporal areas. Sections were hybridized with antisense probes for EphA3 (A,B,D,E), ephrin A5 (F,G,I,J), ephrin A2 (K,L,N,O), or CBF2 (P,Q,S,T). Nasal (anterior) is upwards, temporal (posterior) is downwards. Scale bar: 600 µm. (C,H,M,R,U) Northern blot analysis of E8 chick retina electroporated with CBF1/RCAS. Northern blot analysis was performed using 20 µg of total RNA prepared from the nasal (N) and temporal (T) thirds of E8 retinae transfected with CBF1/RCAS (CBF1). RNA of control retinae (control) was prepared from the left eyes of the same embryos. Probes used for each panel are indicated on the right. A probe for GAPDH, glyceraldehyde phosphate dehydrogenase, was used to indicate the amount of RNA present.

View larger version (65K): [in a new window]   Fig. 4. Misexpression of CBF1 repressing form (CBF1-eve) alters the expression of SOHo1, GH6, EphA3, CBF2 and ephrin A5, but not ephrin A2. (A) Schematic representation of the wild-type and chimeric CBF1. The top drawing represents the wild-type CBF1 protein with myc tag. The repressing form of CBF1 (CBF1-eve) was constructed by fusion of the CBF1 DNA-binding domain (DNA BD) and even-skipped repression domain (eve RD). (B) Nuclear localization of CBF1 proteins. Chick embryonic fibroblasts were transfected with retrovirus vectors for myc-tagged CBF1 (a), or CBF1-eve (b). Nuclear localization of the expressed proteins was visualized by immunofluorescence using anti-myc primary antibody (red). Transfected cells were detected by expression of the viral gag protein from RCAS vector using an anti-gag antibody (green). Scale bar: 20 µm. (C) Whole-mount in situ hybridization of E3 (stage 18-20) chick embryos transfected with CBF1-eve/RCAS using antisense probes for SOHo1 (a), GH6 (b), EphA3 (c) or CBF2 (d). Insets show the normal expression of SOHo1, GH6, EphA3 or CBF2, in the control eyes. Arrowheads indicate the border of endogenous expression areas on the control side. (D) Horizontal section in situ hybridization of E8 retina transfected with CBF1-eve/RCAS using antisense probes for EphA3 (a,b), ephrin A5 (c,d) or ephrin A2 (e,f). The temporal regions of the untransfected retinae (control) are shown in the left panels (a,c,e), and those of the contralateral transfected temporal retinae of the same embryos are shown in the right panels (b,d,f). Scale bar: 100 µm.

View larger version (68K): [in a new window]   Fig. 5. Misexpression of CBF1AA mutant proteins alters expression of SOHo1, GH6, EphA3, CBF2 and ephrin A2, but not ephrin A5. (A) Schematic representation of myc-tagged CBF1AA mutant. The CBF1AA mutant is deficient for DNA-binding activity because of substitutions of asparagine 189 and histidine 193 with alanines. (B) DNA pull-down assays using the nuclear extracts prepared from chick embryonic fibroblasts transfected with myc-tagged CBF1 or CBF1AA (a). Western blot analysis using anti-myc primary antibody indicated the amounts of nuclear extracts used in the DNA pull-down assays (b). (C) Whole-mount in situ hybridization of E3 (stage 18-20) chick embryos transfected with CBF1AA/RCAS using antisense probes for SOHo1 (a), GH6 (b), EphA3 (c) or CBF2 (d). The normal expression of SOHo1, GH6, EphA3 or CBF2 in the control eyes, is shown in insets. Arrowheads indicate the border of the endogenous expression. (D) Horizontal section in situ hybridization of E8 retina transfected with CBF1AA/RCAS using antisense probes for EphA3 (a,b), ephrin A5 (c,d) or ephrin A2 (e,f). The temporal regions of untransfected retinae (control) are shown in the left panels (a,c,e), and those of the transfected ones in the same embryos are shown in the right panels (b,d,f). Scale bar: 100 µm.

View larger version (61K): [in a new window]   Fig. 6. CBF1 inhibits BMP signaling. (A) A luciferase reporter assay using a BMP-responsive reporter construct in the presence of CBF1 or CBF1AA mutant. HEK 293 cells transfected without (-) or with (+) pcDNA/ALK3-CA, Smad1, and Smad4 were measured for luciferase activities in the presence of pcDNA (vector), CBF1/pcDNA (CBF1) or CBF1AA/pcDNA (CBF1AA) by cotransfection. The values are represented by fold induction compared with the basal activity of pcDNA (vector). Data are shown as the mean±s.d. of triplicate experiments. (B) Expression of BMP2 and Ventroptin in the E8 chick retina. Section in situ hybridization was performed with antisense probes for BMP2 (a,b) and Ventroptin (c,d). BMP2 was expressed in a double-gradient pattern that is complementary to Ventroptin. (a,c) Horizontal sections. Nasal (anterior) is upwards, temporal (posterior) is downwards. (b,d) Coronal sections. Dorsal is upwards, ventral is downwards. Scale bars: 600 µm. (e) Schematic representation of the expression patterns of BMP2 and Ventroptin in the retina. (C) Horizontal section in situ hybridization of E8 temporal retina transfected with Ventroptin/RCAS (b) or BMP2/RCAS (d). In the temporal region of a control retina, BMP2 was expressed in the GCL and INL (a). When Ventroptin/RCAS was electroporated, expression of BMP2 was completely repressed (b). In the nasal region of a control retina, Ventroptin was expressed in the INL (c). When BMP2/RCAS was electroporated, expression of Ventroptin was completely repressed (d). Scale bar: 100 µm. (D) Horizontal section in situ hybridization of E8 temporal retina transfected with BMP2/RCAS (b) with a probe for ephrin A2. Ephrin A2 was mainly expressed in the GCL in the nasal region (a). When BMP2/RCAS was electroporated in the retina, expression of ephrin A2 was completely repressed (b). (E) Horizontal section in situ hybridization of E8 temporal retina transfected with CBF1/RCAS (b) or CBF1AA/RCAS (d) with a probe for BMP2. Electroporation of CBF1/RCAS (b) or CBF1AA/RCAS (c) resulted in repression of BMP2 expression in the GCL in the temporal retina of E8 embryos. The temporal region of untransfected retinae (control) are shown in a,c.

View larger version (19K): [in a new window]   Fig. 7. The molecular mechanisms by which CBF1 controls the expression of topographic molecules. (A) Schematic representation of modes of actions of CBF1. Ephrin A5 and ephrin A2 are controlled by CBF1 through transcriptional repression and a DNA binding-independent mechanism, respectively. However, the others are controlled through the dual mechanisms of CBF1. One Ephephrin system is controlled as a set by each mode of CBF1 action. (B) Expressional regulation of asymmetrically distributed molecules along the NT axis by CBF1. CBF1 and Ventroptin repress expression of BMP2 by inhibiting BMP signaling as an interrupter and antagonist, respectively, and induce ephrin A2 expression. However, CBF1 represses the transcription of negative regulators, X and Y. When CBF1 is absent, X downregulates ephrin A5 expression, and Y represses expression of SOHo1 and GH6. When CBF1 is present, X and Y are downregulated, and the expression of ephrin A5, SOHo1 and GH6 is induced. SOHo1 and GH6 inhibit the expression of EphA3. EphA3 and ephrins are directly implicated in the control of axon guidance. CBF1 also represses CBF2 expression. However, downstream target genes of CBF2 have not been identified so far. See text for details.

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