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doi: 10.1242/10.1242/dev.00558

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bozozok directly represses bmp2b transcription and mediates the earliest dorsoventral asymmetry of bmp2b expression in zebrafish

TinChung Leung^1,*, Johannes Bischof¹, Iris Söll¹, Dierk Niessing², Dongyi Zhang³, Jun Ma³, Herbert Jäckle² and Wolfgang Driever^1,

¹ Developmental Biology, Institute Biology 1, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany
² Max-Planck-Institute für Biophysikalische Chemie, Abt. Molekulare Entwicklungsbiologie, Am Fassberg 11, 37077 Göttingen, Germany
³ Department of Developmental Biology, Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA

View larger version (68K): [in a new window]   Fig. 1. A fusion of the Boz-homeodomain with the En repressor domain causes dorsalization, whereas Boz fused to the VP16 activator ventralizes developing embryos. (A-D) An En-Boz repressor domain fusion protein dorsalizes the zebrafish embryo. Top: structure of the En-Boz fusion protein. (A-D) Embryos injected at the one-cell stage with 2 pg En-boz mRNA. Lateral views (A,C) and dorsal views (B,D) of live embryos at 24 hpf. Injected embryos develop a severely dorsalized phenotype. For comparison, see wild-type embryo of same age in (F). (E-H) A VP16-Boz activator-domain fusion protein ventralizes the zebrafish embryo. Top: structure of the VP16-Boz fusion protein. Wild-type embryos at 12-somite stage (E) and 24 hpf (F). (G,H) Embryos were injected at the one-cell stage with 5 pg VP16-boz mRNA. Lateral view at the 12-somite stage (G) and at 24 hpf (H). Injected embryos lack axial mesoderm and develop strong anterior patterning defects including absence of the eye cup (12 som) or eye (24 hpf) and reduced size of the diencephalon.

View larger version (94K): [in a new window]   Fig. 2. Effect of En-Boz repressor and VP16-Boz activator fusion proteins on gsc and chd expression. Wild-type embryos were injected with in vitro transcribed mRNA at the one-cell stage and allowed to develop to 50% epiboly. Expression of chd (A-H) or gsc (I-P) was visualized by in situ hybridization. (A,B,I,J) Uninjected wild-type embryos. chd and gsc are restricted to the dorsal margin of the embryo where the shield will form. (C,D,K,L) Embryos injected with 2 pg boz mRNA. chd and gsc expression expands to the ventral side of the embryo. (E,F,M,N) Embryos injected with 2 pg En-boz mRNA. Similarly, chd and gsc expression expand to the ventral side of the embryo. (G,H,O,P) Embryos injected with 50 pg VP16-boz mRNA. Expression of both chd and gsc at the dorsal margin is strongly reduced. (A,C,E,G,I,K,M,O, lateral views, dorsal at right, animal pole at top; B,D,F,H,J,L,N,P, animal pole views, dorsal at right).

View larger version (82K): [in a new window]   Fig. 3. Control of bmp2b expression by Boz, En-Boz and VP16-Boz. (A-F) Boz and En-Boz can repress bmp2b expression early during gastrulation. Expression of bmp2b at 50% epiboly in wild-type embryos (A,B), embryos injected with 2 pg boz mRNA (C,D) and embryos injected with 2 pg En-boz mRNA (E,F). (G,H) Embryos injected with 50 pg VP16-boz mRNA (right) express increased levels of bmp2b at sphere stage compared with uninjected controls (left). (I) Expression of chd at sphere stage is not affected in embryos injected with 2 pg boz mRNA (right) compared with the uninjected control (left). (J,K) Expression of bmp2b at the sphere stage is severely reduced in embryos injected with 2 pg boz mRNA (right) compared with the uninjected controls (left). (L-O) Comparison of bmp2b expression in wild-type (L,N) and boz mutant embryos (M,O) at sphere stage (red arrowheads mark dorsal margin in L and N, margin in M and O). A,C,E,G,J,L,M: lateral views; B,D,F,H,K: animal pole views; A-H,J-M, dorsal side to the right when the dorsal side could be visually identified; I is an oblique view onto the animal pole from the dorsal side onto the prospective shield region. N and O are enlargements of the dorsal or marginal regions of the embryos pictured in L and M, respectively.

View larger version (48K): [in a new window]   Fig. 4. The effect of Boz on bmp2b transcription is independent of other zygotic gene expression. Expression of bmp2b and gsc at sphere stage in wild-type control embryos (A-F) and in embryos treated with cycloheximide (CHX) between 1.5 hpf and fixation at sphere stage (G-L). Expression of bmp2b (A,G) and gsc (B,H) in uninjected embryos. Expression of bmp2b (C,I) and gsc (D,J) in embryos injected with 2.5 pg boz mRNA. Expression of bmp2b (E,K) and gsc (F,L) in embryos injected with 50 pg VP16-boz mRNA. gsc expression is influenced by boz already at sphere stage (D) and is also used here as a control to determine the effectiveness of cycloheximide treatment (H,J,L).

View larger version (33K): [in a new window]   Fig. 5. Boz binds to control elements in the zebrafish bmp2b gene. (A) Several different end-labeled PCR fragments (F1-F7) covering 3.5 kb of bmp2b genomic DNA, including the first intron, were generated and analysed for the binding of Boz. Further digests of the fragments identified potential binding sites B1-B7, for which electrophoretic mobility shift assays are shown. Only B1 and B4 show efficient Boz binding. The leftmost lanes reveal shift of double-stranded oligonucleotides containing the Bicoid (Bcd) consensus binding site in the presence of Boz. (B,C) The specificity of binding was analysed using various competitor double-stranded oligonucleotides. Putative Boz binding sites B1 and B4 successfully compete both their own and each other's binding to Boz. By contrast, neither oligonucleotide with a Bcd consensus site nor extra bmp2b sites containing TAAT motifs (B2, B3 or B5) can efficiently compete for Boz binding to sites B1 or B4.

View larger version (26K): [in a new window]   Fig. 6. Binding to sites B1 and B4 mediates Boz-dependent repression of bmp2b. Luciferase reporter fusion constructs containing bmp2b sequences from -806 to +2538 fused to the luciferase reporter (BmppLUC) or a control (pCMVtkLUC) were injected into wild-type embryos at the one-cell stage and the amount of luciferase activity was determined at 5 hpf (30% epiboly). The amount of luciferase activity recorded for BmppLUC itself was set as 1. (A) Coinjection of BmppLUC (20 ng µl-1) with boz mRNA or Enboz mRNA (7 ng µl-1) resulted in about 6.5-times repression, whereas co-injection of BmppLUC (5 ng µl-1) with VP16boz mRNA (5 ng µl-1) showed roughly 8.8-times activation. Coinjection of pCMVtkLUC (20 ng µl-1) with boz mRNA or En-boz mRNA (7 ng µl-1) did not have a significant effect on the expression of LUC from the pCMVtkLUC control plasmid. (B) In a separate set of experiments, the contribution of the Boz binding sites B1 and B4 to Boz-dependent repression of BmppLUC was investigated. When injected at a concentration of 20 ng µl-1 together with boz mRNA (7 ng µl-1), BmppLUC showed 10.2-times repression, B1 deletion (Bmp1pLUC) showed 4.6-times repression, B4 deletion (Bmp4pLUC) showed 4.5-times repression and deletions of both B1 and B4 (Bmp1,4pLUC) showed only 1.6-times repression. Thus, most of the Boz-dependent repression of bmp2b appears to be mediated by the sites B1 and B4.

View larger version (13K): [in a new window]   Fig. 7. Role of boz during early dorsoventral patterning. Simplified pathways of regulatory interactions. Solid lines represent interactions during blastula stages, dotted lines represent interactions the significance of which begins during mid-gastrulation. The interactions with vega1 and vega2 are modified from Imai et al. (Imai et al., 2001). Work by Kramer et al. (Kramer et al., 2002) indicates that maternal Smad5 is an ubiquitous activator of bmp2b expression. `X' represents targets of Boz other than bmp2b, which might mediate the function of Boz in anterioposterior patterning. Other effectors of ß-catenin signaling include the probable activators of chd expression not yet identified in zebrafish (i.e. functional homologs of Xenopus Siamois and Twin) as well as Nodal signals, which co-activate gsc through FAST/FoxH1 (Pogoda et al., 2000). Gene names are italicized, protein names begin with capital letters; translation is indicated by gray arrows. Black arrows indicate activation, whereas `T' bars indicate repression.