Figure 1.
Severe Segmentation Defects in Embryos Derived from 2R-14 Germline Clones
Cuticle preparations of newly hatched embryos show three thoracic (T1–T3) and eight abdominal (A1–A8) ventral denticle belts in wt embryos (A). The embryo in (B) is derived from a germline clone homozygous for the original 2R-14 mutant chromosome arm. The phenotype is intermediate to that of gap and pair-rule mutants, with several abdominal denticle belts missing. There is some variation from embryo to embryo with regard to which particular denticle belts are missing, but 100% of the embryos display a segmentation phenotype. Shown in (C) is an embryo derived from germline clones in which the 2R-14 chromosome has been cleaned by recombination. The phenotypes of these embryos are indistinguishable from those derived from the original 2R-14 chromosome. Embryos derived from bks1 germline clones (D) display phenotypes virtually identical to those in 2R-14 mutant embryos.
Figure 2.
Two Bks Isoforms Are Present in Early Embryos
(A) Schematic structure of the two Bks protein isoforms, Bks-A and Bks-B. The N-terminus is rich in serines and glycines, whereas the C-terminus of Bks-B is glutamine- and proline-rich. The D2 domain and the single C2H2-type zinc finger are highly conserved between insects and deuterostomes, whereas high conservation in the D1, D3, and D4 domains is limited to insects. The molecular lesions in the 2R-278 and 2R-339 alleles are indicated.
(B) A wt embryo at the cellular blastoderm stage hybridized with a digoxigenin-labeled antisense bks probe that recognizes both bks isoforms. Due to the maternal contribution, bks transcripts are present ubiquitously in the embryo. Anterior is to the left, and dorsal is up.
(C) RT-PCR experiment demonstrating the presence of both bks-A and bks-B transcripts in early embryos. RNA was isolated from 0–3–h embryos, and primers specific for bks-A or for bks-B were used in the PCR reaction. Products of the expected size were obtained after oligo-dT–primed reverse transcription, but not in the absence of reverse transcriptase.
(D) A wt embryo stained with a Bks antibody raised against the D2 domain [15]. Equal staining intensity is found in all cells of the embryo. In embryos derived from bks278 or bks14 germline clones, nuclear staining is absent, whereas the cytoplasmic (presumably background) staining remains (unpublished data).
Figure 3.
Gap Gene Expression Domains Are Expanded in bks Mutant Embryos
Wild-type (wt) and bks germline clone embryos were hybridized with digoxigenin-labeled RNA probes and are oriented with anterior to the left and dorsal up.
(A–D) Hybridization of a knirps (kni) probe to pre-cellular (A and B) and cellularizing (C and D) embryos. The kni pattern expands greatly towards the posterior in bks14 mutant pre-cellular embryos ([B], see arrow) as compared to wt (A). In cellularizing bks14 embryos (D), the kni pattern remains expanded compared to wt (C), and an ectopic patch occurs in the posterior-ventral part of bks14 embryos (arrowhead in [D]).
(E and F) A kni-lacZ transgene was crossed into wt (E) and bks278 mutant (F) embryos, which were incubated with a lacZ antisense probe. Reporter gene expression expands towards the posterior in bks278 mutant embryos (arrow in [F]).
(G and H) Cellularizing embryos hybridized with a Krüppel (Kr) probe. The central domain of Kr expression present in wt embryos (G) expands in both an anterior and a posterior direction in bks14 mutant embryos ([H], see arrow). In addition, the anterior domain (arrowhead in [H]) is expressed earlier and more broadly than in wt.
(I and J) Hybridization of a giant (gt) probe to cellularizing embryos. Two anterior and one posterior stripe have developed at this stage in wt embryos (I). In bks14 mutant embryos, the gt pattern is variable, but in a vast majority of embryos, there is a delay in the resolution of the anterior gt domain into stripes. In approximately 25% of bks14 mutant embryos, the posterior stripe is reduced or even missing (unpublished data). The embryo in (J) is representative of the majority of bks14 embryos at this stage.
Figure 4.
Tailless (Tll) Repressor Function Is Impaired in bks Embryos
(A–D) Effects of ectopically expressed Tailless (Tll) and Hunchback (Hb) proteins on kni expression in bks mutant embryos. Schematic drawings of the transgenes used to drive ectopic Tll and Hb expression are depicted below the embryo images. (A and B) A snail promoter transgene driving Tll expression in ventral cells was crossed with wt flies or flies containing bks278 germline clones. Expression of tll and kni was visualized in cellularizing embryos by fluorescent in situ hybridization (unpublished data), and by immunohistochemical detection of a digoxigenin-labeled probe, respectively. (A) A wt embryo containing the snail-tll transgene. The posterior kni stripe is repressed in ventral cells (arrow). (B) The posterior kni stripe is not repressed ventrally in a bks mutant embryo containing the sna-tll transgene (arrow). This shows that the repressor activity of ectopic Tll is impaired in bks mutants. (C and D) A hb transgene driven by the snail promoter was introduced into wt embryos or bks14 germline clone embryos. Lateral views of late cellularizing embryos show that ectopic Hb can repress kni expression ventrally in both wt (C) and bks (D) mutants (arrows). Note that the posterior patch of kni expression that occurs in bks mutants is unaffected (star in [D]), presumably because the snail expression pattern does not extend all the way to the posterior.
(E–J) Assay of endogenous Knirps (Kni), Krüppel (Kr), and Giant (Gt) function on reporter transgenes containing synthetic repressor binding sites (schematic drawings of the transgenes are presented below the embryo images). (E and F) Males harboring a lacZ reporter transgene driven by a modified rhomboid NEE enhancer with synthetic Kni binding sites were crossed with wt females or females containing bks14 germline clones. Embryos were collected and hybridized with a lacZ probe. Ventro-lateral views of cellularized wt (E) and bks (F) embryos demonstrate that endogenous Kni protein represses reporter gene expression in both genotypes (arrows). (G and H) Introduction of a modified NEE reporter gene with synthetic Kr binding sites into wt embryos (G) and embryos derived from bks14 germline clones (H). Ventral views of cellularized embryos hybridized with a lacZ antisense probe show that endogenous Kr protein can repress reporter gene expression in both genotypes (arrows). (I and J) Lateral views of a cellularized wt embryo (I) and a cellularized embryo derived from a bks14 germline clone (J) containing a reporter gene with synthetic Gt binding sites, activated by a twist PE enhancer and the rhomboid NEE enhancer, stained with a lacZ probe. Endogenous Gt protein can repress the reporter in both wt and bks mutant embryos (arrows).
Dorsal (dl) and Twist (twi) activators bind the rhomboid and twist enhancers.
Figure 5.
Bks Interacts with Tll and Atrophin
(A) Binding of Bks to Tll in vitro. Left panel shows that in vitro–translated Tll interacts with bacterially produced GST-BksA, but not with GST alone. In the right panel, in vitro–translated Bks-B binds weakly to a GST-Tll fusion protein lacking the DNA binding domain (GST-Tll 101–452), and more strongly with GST-full-length Tll.
(B–E) Genetic interaction of bks with tll mutants. Cellularizing embryos hybridized with a kni probe are oriented with anterior to the left and dorsal up. The kni pattern in wild-type (wt) embryos (B) and embryos from bks278 heterozygous mothers (C) are indistinguishable. In tll1 homozygous embryos (D), the posterior kni domain expands slightly towards the posterior. In tll1 embryos derived from bks278 heterozygous females (E), there is a further expansion of the kni pattern (see arrow).
(F) Bks interacts with the C-terminus of Atrophin. Amino acids (aa) 1,324–1,966 of Atrophin binds the ligand binding domain of Tll, as well as GST-BksA. Truncation of the conserved Bks D2 region (GST-Bks 1–780) does not disrupt binding, but a weaker, independent interaction is found with the D2 domain together with the zinc finger (GST-Bks 834–1,151).
(G) Bks and Tll can be co-immunoprecipitated with Atrophin from Drosophila S2 cells. A stable cell line expressing V5-tagged Bks-B was generated and transiently transfected with FLAG-tagged Tll. Immunoprecipitations with V5, Atrophin, and FLAG antibodies were performed from these cells and compared to normal S2 cells lacking tagged Bks and Tll. The leftmost panel shows a short exposure of a membrane immunoblotted with the V5 antibody, demonstrating the presence of Bks-V5 in transfected cells. The middle panel shows a longer exposure of the same membrane, where Bks-V5 is co-immunoprecipitated with endogenous Atrophin. In the right panel, FLAG-Tll is detected both in the Atrophin and FLAG immunoprecipitates. Arrowheads point to Bks-V5 and Tll-FLAG.
(H) The human Bks homolog ZNF608 (aa 1–600) interacts with aa 600-1191 from human Atrophin-1, showing that the Bks-Atrophin interaction is evolutionarily conserved.
Figure 6.
Bks Associates with the kni and Kr CRMs
(A–D) Chromatin immunoprecipitations (ChIP) were performed on S2 cells or S2 cells expressing V5-tagged Bks-B protein, and associated DNA was quantified by real-time PCR. Mock immunoprecipitation (no Ab), and immunoprecipitation with a negative control antibody (GFP) and with an antibody recognizing the V5 tag (V5) were compared. PCR was performed in triplicate and compared to a standard curve of input DNA. The standard deviation is indicated. (A) An amplicon from the kni CRM is enriched by the V5 antibody in extract from Bks-V5–expressing cells, but not in extract from S2 cells lacking Bks-V5. (B) The V5 antibody precipitates more of Kr CRM DNA from Bks-V5 cells than the control antibody. No enrichment is observed in cells without Bks-V5. (C and D) Bks binding to the kni 5′ UTR (C) or to a locus on Chromosome 4 (D) is similar to the negative controls.
(E) Bks is associated with the kni CRM in early embryos. ChIP followed by real-time PCR was performed on extract from 2–4-h-old embryos with negative control antibody (V5), which was compared to Bks, Atrophin, and Tll antibodies. The Bks, Atrophin, and Tll antibodies precipitated more kni CRM DNA than the V5 control, but similar amounts to V5 of the Chromosome 4 locus.
Figure 7.
Bks Is Capable of Repressing Transcription When Tethered to DNA
(A) The tetracycline repressor DNA binding domain (TetR) was fused to the coding region of bks-A or bks-B. These plasmids were co-transfected with a luciferase reporter gene driven by the actin 5C enhancer (Act enhancer) that contains tet operators (Tet O), as well as an actin 5C-driven lacZ gene to control for transfection efficiency, into mbn-2 cells. Luciferase activity (normalized for ß-galactosidase activity) of unfused TetR is set to 100%, and normalized luciferase activity of TetR-bks fusions plotted relative to the TetR. A schematic drawing of the reporter plasmid is depicted below the histogram.
(B and C) Ventro-lateral view of transgenic embryos expressing lacZ under control of a modified rhomboid NEE enhancer into which Gal4 upstream activating sequences (UAS) have been inserted. LacZ is expressed uniformly in ventral cells in embryos only containing the reporter gene (B). In embryos that additionally express a Gal4 DNA binding domain–BksA fusion protein under control of the Kr CD enhancer (Kr enh), lacZ expression is repressed in the central, Kr expressing domain (C). Schematic drawings of the reporter gene and the Gal4-BksA expressing transgene are shown underneath the embryo images. Dorsal (dl) and Twist (twi) activators bind the modified rhomboid NEE.