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First published online 10 December 2003
doi: 10.1242/dev.00935

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Tcf3: a transcriptional regulator of axis induction in the early embryo

¹ Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10021, USA
² Department of Developmental Biology, Sloan Kettering Memorial Institute, New York, NY 10021, USA

View larger version (32K): [in a new window]   Fig. 2. Generation of Tcf3-/- mice. (A) Targeting strategy. Exons 1-3 of Tcf3 are shown as boxes along the unbroken line representing the Tcf3 gene fragment present in the targeting construct. Broken lines represent Tcf3 gene fragments flanking the targeting construct. The numbers above double-sided arrows represent the size in kb of the fragments generated by digestion with either Bgl2 (B) or HinCII (H). The triangles between exons 1 and 2 and flanking the PGK-NEO cassette represent loxP sites for Cre-mediated recombination. Small single-sided arrows depict the position of forward (for) and reverse (rev) PCR primers used for genotyping of mice. (B) Homologous recombination events in ES clones were confirmed by Southern blotting. +/+, ES cells prior to electroporation; +/neo, one ES clone that had correctly integrated the targeting vector fragment into the Tcf3 locus; +/-, a clone derived from the +/neo clone after electroporation with Cre-recombinase was used to remove the PGKNEO cassette and exon 2 of Tcf3. (C) Genotyping reaction with primers shown in A shows the loss of one copy of exon 2 in Tcf3+/- ES cells and mouse embryos, and the loss of both copies of exon 2 in Tcf3-/- embryos. (D) Western blot of total protein from E8.5 embryos with an antibody directed towards the C terminus of Tcf3 protein confirms loss of Tcf3 protein in Tcf3-/- embryos.

View larger version (69K): [in a new window]   Fig. 1. Activation of Wnt target genes and expression of Tcf3 during gastrulation. (A-E') Tcf/Lef-ß-catenin transcriptional activation revealed by X-gal staining of TOPGal transgenic embryo. The stage of gastrulation (Downs and Davies, 1993) is indicated in the top right-hand corner; the view displayed is indicated in the lower left-hand corner. The direction of posterior (P) to anterior (A) is indicated by an arrow. Boxed regions are those shown at higher magnification. TOPGal activity marks the posterior EEX border at the onset of gastrulation (A) and remains in the primitive streak region throughout gastrulation. (C-E') Neural-fold staged embryos express TOPGal in the primitive and the node. (E,E') Sagittal sections of TOPGal-stained embryos shows expression in both ectodermal and mesodermal cell types and in the posterior cells of the ventral node. (F-I) Indirect immunofluorescence microscopy with antibodies displayed in the lower right-hand corner (color coded according to the secondary antibody used for detection) and embryo sections of the age and plane of sectioning displayed in the upper right-hand corner. (F) Tcf3 (red) is expressed throughout the embryonic ectoderm before primitive streak induction, and (G,H) diminished posteriorly during gastrulation. (I) Close inspection of the node reveals a gradient of Tcf3 expression rising in concentration from the posterior to the anterior of the node. ES, early streak stage; LS, later streak stage; EEX, embryonic/extra-embryonic border; ENF/LNF, early/late neural-fold stage; end, endoderm; mes, mesoderm; ect, ectoderm; ps, primitive streak.

View larger version (71K): [in a new window]   Fig. 3. Morphological defects in Tcf3-/- embryos. (A-F') Wild-type (WT; A,A') and Tcf3-/- (KO; B-F') E8.5 embryos. The view displayed is indicated in the lower left-hand corner. (A,A') Wild-type embryos display bilateral symmetry with two neural-folds (nf) and somites along either side of the AP axis. Rhombomere formation in the neural-folds allows identification of forebrain (fb), midbrain (mb) and hinbrain (hb) regions. The broken line in A' refers to the position of the section displayed in G. (B-D) Mildly affected Tcf3-/- embryos display multiple morphological defects. (B,B') Multiple neural-folds are clearly visible in some mutants. (C) Caudal half of a Tcf3-/- embryo with a secondary neural tube (2°nt) and extra row of somites (asterisks) adjacent to the primary neural tube (1°nt) which runs along the primary AP axis. A higher magnification view of the boxed region in C is displayed in C'. (D) Lack of forebrain structures and an enlarged heart appear in the Tcf3-/- mutant shown. Broken line in D refers to the position of the section displayed in H. (E-F') Ventral views of severely affected Tcf3-/- embryos. (E) Multiple neural groove-like structures (ng) are apparent along the ventral surface of the Tcf3-/- embryo but neural-folds appear to be absent. Red arrowheads indicate areas along the ventral surface that appear to lack underlying mesoderm and ectoderm. The boxed region in F is shown in higher magnification in F'. (G,H) Toluidine Blue stained semithin sections of embryos in A' and D, respectively. Notice the single neural-groove in G and the three neural grooves in H. (I) E7.5 wild-type or knockout embryos, as indicated at the top of each embryo. A Toludine Blue stained semithin section from the plane indicated with the broken line is displayed below each embryo. Notice the morphological defects in the primitive streak region of Tcf3-/- embryos manifest either a bulge (arrowheads, middle embryo) or a second primitive streak region (arrowheads, right embryo). ncd, notochord; ne, neurectoderm; fg, foregut, EEX, embryonic/extra-embryonic border.

View larger version (98K): [in a new window]   Fig. 4. Tcf3-/- embryos display defective patterning of anterior neural regions. In situ hybridization of E8.5 wild-type (WT; A,A',C,C',E,E') and Tcf3-/- (KO; B,B',D,D',F,F') embryos using cRNA probes as indicated. Wild-type embryos display prominent staining for Hesx1 (A,A') in the forebrain whereas mutant embryos (B,B') display very weak Hesx1 staining. (C,C') En1 is expressed in the midbrain-hindbrain boundary caudal to the midbrain. The distance of expression to the rostral end of the embryo is marked by the curved bracket (C). (D,D') En1 expression is observed in the mutant embryos, but the distance from the rostral end is diminished (bracket). Note also the deformed neural groove in mutant (D'). (E,E') Robust Krox20 is present in rhombomeres 3 and 5 of the wild-type embryo. (F,F') Weak Krox20 expression is observed in only one stripe of neurectoderm in the mutant embryos. In addition, the expression of Krox20 is expanded laterally compared with the pattern seen in the wild type.

View larger version (112K): [in a new window]   Fig. 5. Duplications and expansions of node and notochord in Tcf3-/- embryos. (A-B'') Scanning electron microscopy (SEM) images of the ventral surface of wild-type (WT; A,A') and Tcf3-/- (KO; B-B'') embryos (anterior towards the lower left-hand corner). Arrows: Green, nodes; blue, notochords; red, endodermal strip. White line indicates notochordendoderm boundary in B and B'. White boxes in A and B outline areas magnified in A' and B', respectively. Note the club-like structure of the single node and notochord in the wild-type embryo and the duplicated node and expanded notochord in mutant embryo. (C-F') Whole-mount in situ hybridizations of E8.5 wild-type and knockout embryos, probed with digoxigenin-labeled cRNAs for brachyury or Foxa2 as indicated. Anterior is towards the top of each image. The three embryos in each panel in D and F show different representative aberrant patterns, reflective of the extent of node/notochord multiplication. Opposing arrowheads indicate thickness of notochords (ncd); arrows indicate splitting of the notochord, often seen in mutant embryos. (G,G') Tcf3-/- embryo with a rostral extension probed for brachyury expression. Anterior is leftwards for both images. The primary primitive streak (1°PS) and a secondary primitive streak (2°PS) are positive for brachyury expression. Emerging from the secondary primitive streak are structures similar to a node (arrows) and notochord (arrowheads).

View larger version (113K): [in a new window]   Fig. 6. Mesodermal patterning defects in Tcf3-/- embryos. (A-L') In situ hybridization with wild-type (WT) or Tcf3-/- (KO) E7.0 embryos using the probes indicated in the upper right of each panel. The view displayed is indicated in the lower right corner and embryos are positioned as indicated in the key EEX marks the embryonic/extra-embryonic border. (A-D) Endodermal markers Cer1 (A-B') and Hex (C,D) are maintained in Tcf3-/- (B,B',D) embryos. (E-F') Brachyury marks the primitive streak and axial mesendoderm in the wild-type (E), and it is maintained in Tcf3-/- embryos (F,F') but adopts a bent or twisted pattern (arrows) consistent with multiple primitive streaks. (G-H') Cripto expression is similar in wild-type (G) and Tcf3-/- embryos (H,H'). (I-J') Foxa2 expression marks the axial mesoderm of the APS in the wild type (I). In Tcf3-/- embryos, Foxa2 expression is either expanded (J) or ectopically expressed such as shown circumferentially along the EEX border in J'. (K) Lefty2 is expressed in the lateral mesoderm and its pattern reveals a `notch' (outlined in red) at the distal tip of the wild-type embryo, reflective of the axial mesoderm-mesendoderm. (L,L') In Tcf3-/- embryos, Lefty2 expression is diminished and reveals a larger `notch' (outlined in red) indicating the increased axial mesoderm in the mutant.

View larger version (84K): [in a new window]   Fig. 7. TOPGal expression patterns maintained in Tcf3-/- embryos. TOPGal transgenic, wild-type (WT) or Tcf3-/- (KO) embryos of the indicated stages of development were stained with X-gal to identify the location of activation of Lef/Tcf-ß-catenin target genes. (A) Lateral view of a wild-type late streak embryo is weakly positive for TOPGal activity in the primitive streak region. (B,C) Tcf3-/- embryos display TOPGal positive staining in the primitive streak region. The intensity of staining is indistinguishable from wild type. (D,D') Headfold staged Tcf3-/- embryos display a TOPGal expression pattern indistinguishable from wild-type embryos (see Fig. 1C,D) and include positive TOPGal expression in the node. (E) Tail of a wild-type E8.5 embryo. TOPGal is present in the posterior node and the mesenchymal cells near the node. (F) Tcf3-/- E8.5 embryo. TOPGal expression is found in the tail of the embryo. (F') Dorsal view shows a duplication of the node in this embryo. TOPGal is maintained in the same pattern as wild type in the node and the surrounding mesenchyme, despite the duplication.