spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tallafuß, A.
Right arrow Articles by Bally-Cuif, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tallafuß, A.
Right arrow Articles by Bally-Cuif, L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The zebrafish buttonhead-like factor Bts1 is an early regulator of pax2.1 expression during mid-hindbrain development

Alexandra Tallafuß1,2, Thomas P. Wilm2,*, Michèle Crozatier3, Peter Pfeffer4,{ddagger}, Marion Wassef5 and Laure Bally-Cuif1,2,§

1 Zebrafish Neurogenetics Junior Research Group, Institute of Virology, Technical University-Munich, Trogerstrasse 4b, 81675 Munich, Germany
2 GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, Ingolstaedter Landstrasse 1, 85764 Neuherberg Germany
3 Centre de Biologie du Développement, UMR 5547 CNRS/UPS, 118 route de Narbonne, 31062 Toulouse, France
4 Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria
5 CNRS UMR 8542, Ecole Normale Supérieure, 46 rue d’Ulm, 75005 Paris, France
* Present address: Vanderbilt University, Department of Biological Sciences, Box 1634 Station B, Nashville TN, 37235 USA
{ddagger} Present address: AGResearch P/Bag 3123, Hamilton, New Zealand.



View larger version (30K):

[in a new window]
  		Fig. 1. Structure of the Bts1 protein. (A) Zinc finger domains of the 11 zebrafish Btd/Sp-family members (Bts proteins) isolated, aligned with the corresponding domains of Drosophila Btd (Wimmer et al., 1993) and mouse Sp1 (Kadonaga et al., 1987). Positions of the primers used in the degenerate PCR reaction are indicated (arrows). Each zinc finger has the structure 3x(C2H2) (red boxes highlight Cys and His residues) and is preceded by a ‘Btd box’ (boxed in black for Btd, mouse Sp1 and Bts1, not indicated for others). The Cys doublet mutated in the negative control-construct Bts1C->T (see Fig. 5) is boxed in blue. The expression profile of each bts gene at the tail bud stage is summarized in the right column. jct, junction; gl, gland; ND, not determined; NT, neural tube; olf, olfactory placodes; TB, tail bud; ves, vesicle. (B) Sequence of the Bts1 protein. The zinc-finger domains are in red and the Btd-box is boxed in black. S/T and Q-rich, potential transcriptional activation domains are, respectively, in green and blue. The N-terminal domain resembling that of Sp1, Sp2, Sp4 and Sp5 is underlined. (C) Structural alignment of Bts1 and other Btd/Sp proteins (Kadonaga et al., 1987; Hagen et al., 1992; Wimmer et al., 1993; Wimmer et al., 1996; Supp et al., 1996; Harrison et al., 2000). Percentages of similarity between Bts1 and other proteins are given for the zinc finger/Btd box (red/black). Q-rich domains are blue (the Q domain of Bts1 only resembles that of Btd (dark blue) but does not align with others (light blue)). S/T-rich domains are green and the N-terminal domain grey. The transcriptional activation domains identified in Sp1, Sp3 and Sp4 are labelled A-D.

 


View larger version (129K):

[in a new window]
  		Fig. 2. Bts1 is a transcriptional activator in vivo. Expression of collier (col) revealed by in situ hybridization at the head-trunk junction of the Drosophila blastoderm in wild-type embryos (A) and in btd mutant embryos carrying one copy of bts1 under control of btd regulatory elements (B). btd mutants show no expression of col (not shown). bts1 can partially rescue col expression in btd mutants, in a correct spatiotemporal manner.

 


View larger version (78K):

[in a new window]
  		Fig. 3. Expression of bts1 during gastrulation and early somitogenesis, as revealed by whole-mount in situ hybridization at the stages indicated (% of epiboly). (A-F) Dorsal views, anterior towards the top; (G-J) sagittal views, anterior towards the left. Open arrowheads indicate the blastoderm margin, black arrowheads the mid-hindbrain domain, and small arrows point at hypoblastic expression. (A',C',D',D'') Sagittal sections at the levels indicated, dorsal towards the right, anterior towards the top. bts1 expression is first detected at 30% epiboly (A,A') along the ventral and lateral margins of the blastoderm (arrows) and in the yolk syncitial layer (small arrow). During gastrulation (B-F), expression is maintained in the posterior epiblast up to a sharp limit at the mid-hindbrain level, and in hypoblast cells bordering the prechordal plate (small arrow). From the end of gastrulation (F-H), bts1 expression is confined to the mid-hindbrain level and tail bud and extinguishes from the rest of the epiblast. Additional expression sites during later somitogenesis (I,J) include the otic vesicle, somites and diencephalon.

 


View larger version (58K):

[in a new window]
  		Fig. 4. Comparison of bts1 expression with other mid-hindbrain markers. Whole-mount in situ hybridization was performed at the 75% epiboly (A-E), tail bud (G-K) and five-somite (M-Q) stages with the probes indicated (colour-coded) (dorsal views, anterior towards the top). (A,B) Single staining for bts1 and fkh3, respectively (whole-mount views of half embryos) (arrow in A indicates anterior limit of bts1; bracket in B indicates ‘diencephalic wings’ of fkh3 expression). (D) Bright-field view of a flat-mounted MHD, all other panels show a bright field view (left, red and blue labelling) and the contralateral fluorescence view (right, red labelling only) of flat-mounted neural plates. (F,L,R) Corresponding schematics of genes expression profiles (including data not shown) at 75% epiboly, tail bud and five somites, respectively. Note that anteriorly, bts1 expression never extends to the presumptive diencephalon (compare A with B), and that it crosses the caudal border of otx2 expression at all stages.

 


View larger version (80K):

[in a new window]
  		Fig. 5. Bts1 is sufficient to induce pax2.1 expression in the anterior neural plate. pax2.1 (A-D) or pax2.1 and gsc (E,F) expression revealed by whole-mount in situ hybridization (blue staining) at the tail bud stage on embryos injected with (A,B,D-F) wild-type bts1 RNA, or (C) mutant bts1C->T RNAs, as indicated (bottom left of each panel). (A-C) High magnifications of the MHD in flat-mounted embryos, anterior towards the top. (E) A whole-mount view, anterior towards the left. (D,F) Sections of the embryos in A,E (respectively) at the levels indicated, anterior towards the left. The red arrows in E,F point to gsc expression, and the broken line in F delimits the anterior mesendoderm/neural plate border. All injections were made into in one central blastomere of the 16-cell embryo, leading to a mosaic distribution of the injected RNA in the presumptive neural plate (see expression of the ß-galactosidase tracer (brown nuclei) and in particular D,F). Misexpression of bts1 induces ectopic pax2.1 expression (black arrows in A,B,E, black bars in D,F) anterior to the MHD (endogenous pax2.1 expression is indicated by the white arrowheads or white bars), in broad patches (A,D-F) or in scattered cells (B). Mutant bts1 RNAs (C, and data not shown) have no effect.

 


View larger version (121K):

[in a new window]
  		Fig. 6. Bts1 is necessary to the expression of pax2.1 and its dependent cascade in the MHD. (A-D) pax2.1 expression revealed by whole-mount in situ hybridisation (purple) at 90% epiboly (B) or tail bud (A,C,D) after injection of MObts1 (A-C) or the mismatch control MObts1{Delta}4 (D). All injections were made at the 16-cell stage into one central blastomere. (C) A biotinylated control MO of unrelated sequence co-injected as a tracer (turquoise staining) to monitor the exact distribution of targeted cells (turquoise arrows) compared with pax2.1-expressing cells (purple arrows); the area shown is a high magnification of the domain indicated by the black arrow in the inset. (D) nls-lacZ RNA used as a tracer to reveal the targeted area (brown staining). (A) Whole-mount views; (B-D) flat-mounts, anterior towards the top; arrows point to injected areas (affected and unaffected expression are indicated by filled and open arrows, respectively). Note that the injection of MObts1, but not MObts1{Delta}4, strongly diminishes the number of pax2.1-positive cells from the onset of pax2.1 expression (B), and that cells maintaining pax2.1 expression have not been targeted by the injection (C). (E-H) Expression of her5 (E, tail bud), fgf8 (F, tail bud), wnt1 (G, one to two somites) and eng2 (H, three somites) upon injection of MObts1 (conditions as in D). Note that these expression are unaffected. (I-N) Expression of pax2.1 (I,J), eng3 (K,L) and pax5 (M,N) at the five-somite stage upon injection of control MO or MObts1, as indicated. (I,J) Dorsal views, anterior towards the top; (K-N) Optical coronal sections, dorsal towards the top. Note that at five somites, the pax2.1-dependent markers eng3 and pax5 are also affected.

 


View larger version (103K):

[in a new window]
  		Fig. 7. bts1 expression at the MHD during gastrulation requires Fgf8 and is activated by Wnt signalling. (A,B) Whole-mount dorsal views of bts1 expression at the tail bud stage, anterior to the top, without (A) or after (B) treatment with the inhibitor of Fgf signalling SU5402 between the stages dome and tail bud. Note the strong reduction in expression at the mid-hindbrain in B (arrowhead), while expression at the blastoderm margin is not affected (white arrow). (C,D) Flat-mounted views of bts1 expression in the mid-hindbrain area at the 90% epiboly stage, anterior towards the top, in wild-type (+/+) (C) versus ace homozygous mutants (D), as indicated. Mid-hindbrain expression of bts1 is strongly reduced and maintained only laterally (arrowheads); it remains unperturbed at the blastoderm margin (white arrow). (E,F) bts1 expression in embryos grafted with wnt1-expressing cells within the anterior neural plate. Endogenous bts1 expression at the MHD is indicated by the arrowhead. Grafted cells were co-injected with nls-lacZ RNA and are visualized by anti-ß-galactosidase immunocytochemistry (brown nuclei). (F) A high magnification of the grafted area (boxed in E). bts1 expression is induced around wnt1-expressing cells.

 


View larger version (88K):

[in a new window]
  		Fig. 8. bts1 expression during somitogenesis distinguishes Pax2.1 and Fgf8 functions. (A-D) Comparison of bts1 and pax2.1 expression in wild-type (left) or ace mutant (right) embryos at the 13-somite stage. The MHD is indicated by the arrowhead. bts1 expression is unperturbed in ace (B), when most pax2.1 expression has already been eliminated (D). (A,B) ace embryos identified by their reduced otic vesicles, which also express bts1 (not visible on the figure). (E-H) Comparison of bts1 and fgf8 expression in wild-type (left) or noi mutant (right) embryos at the 10-somite stage. bts1 expression is strongly diminished following the same schedule as other MHD markers (e.g. fgf8). lim5 expression (red) is unperturbed.

 


View larger version (12K):

[in a new window]
  		Fig. 9. A model of MHD induction incorporating Bts1 function. Evidence in all vertebrates suggest that the expression of early MHD markers (her5, pax2.1, wnt1 and fgf8) (green) is established by following independent pathways. Bts1 (red) is a selective inducer of pax2.1 expression, and its own expression depends on Fgf8 signalling (blue). Other factors regulating bts1 expression might include Wnt molecules. In turn, Pax2.1 induces eng3 and pax5. Bts1 might also directly regulate the expressions of eng3 and pax5 (red arrows).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2001