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First published online 14 February 2007
doi: 10.1242/dev.000265

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Hippocampus-like corticoneurogenesis induced by two isoforms of the BTB-zinc finger gene Zbtb20 in mice

Molecular Neurobiology Laboratory, Medical Biotechnology Center, University of Southern Denmark, J. B. Winslowsvej 25, DK-5000 Odense C, Denmark.

View larger version (89K): [in this window] [in a new window]   Fig. 1. Zbtb20 misexpression in dorsal telencephalon of transgenic mice. (A) Schematic illustration of the Zbtb20S and Zbtb20L recombinant constructs used for pronuclear microinjection. (B) Western blot analysis of Zbtb20S and Zbtb20L expression in the hippocampal formation of wild-type mice and mice harboring D6/Zbtb20S (TgS), D6/Zbtb20L (TgL) and D6/Zbtb20S//D6/Zbtb20L (TgS/L) transgenes (left). Western blot analysis of postnatal Zbtb20 expression in the hippocampal formation of TgS and wild-type littermates (right). (C,D,F,G) Indirect immunofluorescent detection in wild-type and TgS E16 and E18 coronal hemisections of Zbtb20 protein, counterstained with DAPI. (E,H) Horizontal sections of P5 wild-type and TgS hippocampal formations showing co-expression of Zbtb20 (red) and NeuN (green) by indirect immunofluorescence. CA1 and CA3 indicate hippocampus; CP, cortical plate; DG, dentate gyrus; Hip, presumptive hippocampus; LV, lateral ventricle; Sub, subiculum; WT, wild type.

View larger version (96K): [in this window] [in a new window]   Fig. 2. Delayed radial migration of immature projection neurons in Zbtb20 transgenic mice. (A,G) Nissl-stained wild-type and Zbtb20 transgenic E18 coronal sections of medial-dorsal telencephalon. Compared with wild type (A), Zbtb20 transgenic embryos harbor streams of radial migrating cells in the intermediate zone (arrow in G). (B-F,H-L) Cell birth date and cortical migration analyses by indirect immunofluorescent detection of BrdU-labeled cells (arrows in B,C,H,I) and by fluorescent visualization of EGFP-labeled neurons in CP (arrows in D-F,J-L) at late P0 (D,E,J,K) and P14 (F,L) following in vivo transfection with a pCIG2-EGFP expression vector at E15, in coronal sections counterstained with DAPI. (M) Distribution of EGFP-labeled cells in P0 brains transfected with pCIG2-EGFP at E15.The data are presented as means±s.e.m. *, P=0.021, **, P=0.029, ***, P0.001. Deep, deep half of CP; Superficial, superficial half of CP.

View larger version (166K): [in this window] [in a new window]   Fig. 3. Cortical lamination defects and hippocampus-like cytoarchitectonic transformations. (A-K) Nissl-stained adult wild-type, TgS/L, TgS and TgL coronal forebrain sections at rostral (A,D,G), mid (B,E,H) and caudal axial levels, in low (C,F,I) and high (J,K) magnification views. Note the compact hippocampus-like pyramidal cell layer in subicular (Sub) and retrosplenial areas (Rsc) in TgS, TgS/L and TgL brains (arrows in F,I,K). (L,M) Nissl-stained adult wild-type and TgS transgenic horizontal sections showing that the subicular pyramidal cell layer in the Zbtb20 transgenic mice is transformed into a compact hippocampus-like pyramidal cell layer in TgS (arrow in M). aq, aqueduct of Sylvius; cc, corpus callosum; Cgc, cingulate cortex; DG, dentate gyrus; M, motor cortex; S, somatosensory cortex; Str, striatum (caudate putamen); 3V, third ventricle; V, visual cortex.

View larger version (77K): [in this window] [in a new window]   Fig. 4. The transformed retrosplenial cortex is composed of large hippocampus-like pyramidal neurons. (A,E-G) Golgi impregnation of wild-type (A) and TgS transgenic (E-G) posterior Rsc revealing a CA1-like morphology of pyramidal neurons. (B-D,H-J) Alterations in pyramidal neuron morphology in TgS transgenic (H-J) posterior Rsc compared with wild type (B-D) shown by fluorescent visualization of EGFP-labeled outer layer neurons at P14, following in vivo transfection with a pCIG2-EGFP expression vector at E15, in coronal sections counterstained with DAPI (B,H). Note that Rsc pyramidal neurons born after E15 in TgS transgenic brains are large cells that settle in a compact pyramidal cell layer (J), whereas these neurons are small outer-layer pyramids in wild-type brains (D).

View larger version (114K): [in this window] [in a new window]   Fig. 5. Altered pattern of molecular marker expression in Zbtb20S transgenic cortex. (A,G) Low-magnification views of indirect immunofluorescent detection of Oct-6-labeled cells in coronal sections of P14 wild-type (A) and Zbtb20 transgenic (G) posterior cerebral cortex. (B,H) Higher-magnification views of A and G with focus on CA1, Sub and Rsc. (C,I) Immunohistochemical detection of ER81-labeled cells in coronal sections of P14 wild-type (C) and Zbtb20 transgenic (I) CA1, Sub and posterior Rsc. (D,E,J,K) Indirect immunofluorescent detection of Brn-1- and Brn-2-labeled cells in coronal sections of P0 wild-type (arrows in D and E) and Zbtb20 transgenic (J,K) posterior cerebral cortex. (F,L) High-magnification views of indirect immunofluorescent detection of reelin (arrows) in retrosplenial cortex of coronal hemisections of P0 wild-type (F) and Zbtb20 transgenic (L) brains. Insets (F,L), low-magnification views of indirect immunofluorescent detection of reelin in posterior cerebral cortex. (M-P) Indirect immunofluorescent detection of Brn-1 and Brn-2 expression in sub-pial nodules (arrows in N and P) of lateral areas of Zbtb20 transgenic visual cortex, counterstained with DAPI (M,O).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Zbtb20S transgenic mice display behavioral abnormalities. (A) Visual cliff measures of percentage of steps to safe side in age and sex-matched (females) wild-type (n=11) and Zbtb20S (n=13) mice. *P0.001. (B-D) Circular platform maze performed on the mice shown in (A). (B) Latency to enter the escape hole on each trial day. (C) Number of errors committed prior to locating the escape hole on each day of the trial. (D) Number of probes to the learning period escape hole (L) compared with approaches to all holes including the new reverse learning period escape hole (R) at the first trial day of the reverse learning period. All data are presented as means±s.e.m.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Model. (A) Multipotent cortical progenitors harbor a cell-autonomous program in which, by asymmetric divisions, they become progressively restricted in developmental potential. At an early stage of corticoneurogenesis (around E10 in mice) they produce reelin-positive CR neurons followed by deep-layer and outer-layer pyramidal neurons and eventually macroglia [modified from Mizutani and Gaiano (Mizutani and Gaiano, 2006)]. The transcription factor Foxg1 appears to function as a constitutive active molecular switch required for early fate transitions in asymmetric proliferating precursors (i.e. by suppressing the production of reelin-positive CR neurons). (B) Ectopic expression of Zbtb20 in immature cortical pyramidal neurons leads to hippocampus-like neurogenesis of these cells apparently without affecting the early generation of CR neurons. The hippocampus-like transformations involved a deficiency in neurons expressing deep-layer (i.e. ER81) and outer-layer markers (i.e. Brn-1 and Brn-2) in Zbtb20 transgenic brains. The model suggests that Zbtb20 represses cell fate transitions in newborn pyramidal neurons and orchestrates the invariant morphogenesis of these neurons. The differentiation of macroglia from late precursors was not investigated in this work.

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