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First published online 4 October 2006
doi: 10.1242/dev.02600

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COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth

TIGEM (Telethon Institute of Genetics and Medicine), Developmental Disorders Program, Via P. Castellino 111, 80131 Napoli, Italy.

View larger version (74K): [in a new window]   Fig. 1. Expression and inactivation of COUP-TFI in the developing forebrain. (A-J) Coronal sections immunostained for COUP-TFI at the ages indicated above. A to D are consecutive rostral to caudal sections. (E) Enlargement of the presumptive cingulate cortex in which COUP-TFI is present in scattered cells (arrows). (F) Detail of one half of the cingulate cortex, double immunolabelled for COUP-TFI (green) and Reelin (red), shows that COUP-TFI-positive cells do not co-localize with the marginal zone marker Reelin. (G-J) Note how COUP-TFI starts to be expressed in the dorsal thalamus (dTh) from the onset of thalamic differentiation and is maintained at later stages in distinct nuclei. (I) In the hippocampus high levels of COUP-TFI are present in the hippocampal plate and in the intermediate zone. Scattered cells are also detected in the inner marginal zone. (J) Detail of the dorsal thalamic region in a section posterior to (I) shows high expression of COUP-TFI in the lateral geniculate and ventroposterior nuclei. Dashed lines demarcate the LGN and VP thalamic nuclei. The zona incerta is also positive for COUP-TFI. (K) Diagram showing the gene targeting strategy used to obtain the COUP-TFInull allele after Cre-recombination in embryonal stem cells. The lox P sites are depicted as triangles. Blue boxes represent exons. Appropriate restriction enzyme sites are indicated. Red lines indicate the expected fragment size after EcoRI digestion of COUP-TFIwt, COUP-TFIfloxneo and COUP-TFInull genomic DNA, as shown in a Southern blot (L). (M) PCR genotyping on wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mice using the primers indicated in K as black arrows. (N) Western blot shows total absence of COUP-TFI protein in homozygous mutant animals. (O) Coronal sections of wild-type and homozygous E15.5 embryos confirm complete absence of COUP-TFI protein in homozygous brains. Scale bars: 200 µm. aa, anterior amydgaloid area; aon, anterior olfactory nucleus; BG, basal ganglia; cam, claustro-amygdaloid complex; cp, cortical plate; hip, hippocampus; hp, hippocampal plate; ic, internal capsule; imz, inner marginal zone; iz, intermediate zone; LGN, lateral geniculate nuclei; pir, piriform cortex; rms, radial migratory stream; s, septum; sp, subplate; VP, ventroposterior nuclei; ZI, zona incerta.

View larger version (57K): [in a new window]   Fig. 2. Abnormal development of the corpus callosum and hippocampal commissure in COUP-TFInull brains. Coronal sections through the telencephalon of wild-type and COUP-TFInull embryos at the stages indicated and immunostained for calretinin (A,A') and L1 (B-E'). (A-B') At E18.5 corpus callosum fibres cross the midline in wild-type embryos (A,B), but remain ipsilateral in null mutants (A',B'). Note how the cingulate cortex and the cingulum bundle are ventrally extended in the null mutants (arrows in A',B'). This phenomenon is quantified in F, where the length of the cingulate cortex is compared to the total DV length of the forebrain. (C'-E') Serial rostrocaudal sections of E18.5 embryos indicate a milder phenotype in 3/19 null mutants. Arrowheads in C',D' point to partial crossing. (E') In more caudal sections the mutants lack a morphologically distinct corpus callosum and hippocampal commissure (arrow). The arrowheads in the insets C'-E' indicate defasciculated fibres stopping abruptly at the midline. Scale bars: 200 µm. cb, cingulum bundle; cc, corpus callosum; fo, fornix; hc, hippocampal commissure; Lc, length of the cingulate cortex; Lt, total DV length of the forebrain; NULL, COUP-TFInull embryos; WT, wild-type embryos.

View larger version (80K): [in a new window]   Fig. 3. Aberrant projections of the corpus callosum and hippocampal commissure in COUP-TFInull brains. Coronal sections through the telencephalon of E18.5 wild-type and COUP-TFInull fetuses in which DiA was implanted in the rostromedial neocortex (A,A') and DiI in a more caudomedial position (B,B'). The inset in A indicates the positions of the dyes. Note the presence of Probst bundles (arrowheads in A',B') in the ipsilateral cortex. (D-E') Consecutive rostrocaudal sections, in which DiI was inserted in the hippocampus, show how the hippocampal commissure fails to cross in null mutants (arrows in D',E'). At all levels, labelled fibres do not cross the midline but turn ventrally. The white arrowheads in D and E indicate some retrogradely labelled neurons in the medial septum/diagonal band complex. Scale bars: 200 µm. NULL, COUP-TFInull fetuses; Pb, Probst bundles; WT, wild-type fetuses.

View larger version (91K): [in a new window]   Fig. 4. Abnormal axon trajectories in the AC of COUP-TFInull brains. Serial free-floating coronal (A-D') and horizontal (G-I') sections through the telencephalon of E18.5 wild-type and COUP-TFInull fetuses immunostained for L1. In wild-type embryos, the anterior tract of the AC is visible as distinct spots in anterior coronal sections (A,B) and as a horseshoe-shaped tract in horizontal sections (H). The posterior tract is positioned more dorsally than the anterior one and forms a laterally directed tract connecting the two temporal lobes (G). The two tracts show decussation jointly at the midline (C,D,G). In the COUP-TFInull brains, the anterior and posterior tracts form ectopic fascicles that innervate the rostromedial cortex (arrows in H',I'). The main tract shows decussation at the midline but its overall diameter is larger (arrowheads in C',D',G',H',I'). The arrows in A' and B' indicate ectopic fascicles of the anterior tract in rostral sections. (E-F') Double dye tracing of E18.5 brains in which a crystal of DiI was placed in the hippocampus (E,E') and a crystal of DiA was placed in the anterior branch of the AC (F,F'). Note that in the null embryos hippocampal-septal projections enter the AC tract (arrowhead in E') and the AC fibres project dorsally towards the hippocampal commissure (arrowhead in F'). (J) Quantification of the thickness of wild-type (blue) and null (red) AC measured at the midline. In the null embryos, the AC is significantly larger in both the coronal and horizontal planes. Scale bars: 200 µm. acA, anterior tract of the AC; acP, posterior tract of the AC; NULL, COUP-TFInull fetuses; WT, wild-type fetuses.

View larger version (27K): [in a new window]   Fig. 5. Altered gene expression and protein levels of actin and microtubule cytoskeleton molecules in COUP-TFInull. (A) Fold-change variations (expressed as 2-Ct values) in MAP1B, MAP2, Rnd2 and CAP1 mRNA expression levels between the wild-type and COUP-TFInull E14.5 brains after QRT-PCR. The data are normalized to levels of expression of all of the genes in wild-type embryos as 1.0. Significant differences are indicated by asterisks. (B) Western blots of E14.5 brain extracts from wild-type and COUP-TFInull littermates. The blots were incubated with the antibodies listed on the right-hand side. (C-E') Coronal sections of E15.5 wild-type and COUP-TFInull dorsolateral cortices immunostained for MAP1B, RND2 and TAU antibodies. (C',D') The arrows indicate reduced MAP1B and increased RND2 protein levels in the upper intermediate zone, respectively. The asterisks show altered expression in the marginal zone and the arrowhead in D' points to an expanded expression of RND2 into more dorsal regions. (E,E') The arrowheads show no changes in expression levels of the general microtubule-associated protein TAU. Scale bar: 200 µm. IZ, intermediate zone; MZ, marginal zone; NULL, COUP-TFInull fetuses; VZ, ventricular zone; WT, wild-type fetuses.

View larger version (76K): [in a new window]   Fig. 6. Altered general morphology and morphometric parameters of COUP-TFI-deficient primary neurons. (A-G'') All the neurons were labelled with an antibody against tyrosinated -tubulin (green) and rhodamine-phalloidin for actin (red). (A''-G'') Merged figures in which blue staining (DAPI) reveals the nucleus. (A-B'') After 12 hours of culture, COUP-TFInull neurons show an abnormal distribution of actin filaments (arrows in B') and no or very little protrusions (arrowheads in B''). (C) Graph showing the percentage of neurons that display a spherical phenotype at different hours after plating. (D-E'') After 24 hours in culture, COUP-TFInull neurons have extended some neurites; however, they present a curled and abnormal growth pattern (arrow in E''). The arrowheads in E'' indicate the ectopic presence of protrusions. (F-G'') After 48 hours in culture most of the COUP-TFInull neurons do have a more elongated axon; however, these neurons display numerous short filopodial extensions (arrowheads in G''), and axons tend to curl abnormally (arrow in G''). The red arrowhead indicates abnormal accumulation of tubulin- and actin-rich structures around the nucleus. Scale bars: 10 µm in A-B''; 20 µm in D-G''. NULL, COUP-TFInull neurons; WT, wild-type neurons.

View larger version (48K): [in a new window]   Fig. 7. Decreased MAP1B and increased RND2 levels in axons of COUP-TFI-deficient primary neurons. (A-B'') Confocal images of primary hippocampal neurons after 24 hours in culture labelled with antibodies recognizing MAP1B and TAU. MAP1B and TAU staining overlap in the cell body and neurites in wild-type neurons (A-A''). Axonal MAP1B levels are clearly reduced in COUP-TFInull mutants, as shown by the arrowheads in B and in a merged image (B''), whereas TAU fluorescence levels do not change (B'). (C) Quantification of TAU and MAB1P fluorescence in the cell body and in the axon via relative intensity measurement (see text for more details). (D-E'') Immunofluorescence of RND2 and TAU in primary neurons after 48 hours in culture. RND2 is restricted to the cell body close to where the axon emerges in wild-type neurons, but is upregulated and ectopically distributed along the whole length of NULL neurons, as indicated by arrowheads (E-E''). Scale bars: 20 µm. NULL, COUP-TFInull neurons; WT, wild-type neurons.

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