Brickman JM et al. (2000), Hex is a transcriptional repressor that contrib...

XB-ART-11037

Development 2000 Jun 01;12711:2303-15. doi: 10.1242/dev.127.11.2303.

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Hex is a transcriptional repressor that contributes to anterior identity and suppresses Spemann organiser function.

Brickman JM , Jones CM , Clements M , Smith JC , Beddington RS .

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One of the earliest markers of anterior asymmetry in vertebrate embryos is the transcription factor Hex. We find that Hex is a transcriptional repressor that can be converted to an activator by fusing full length Hex to two copies of the minimal transcriptional activation domain of VP16 together with the flexible hinge region of the (lambda) repressor (Hex-(lambda)VP2). Retention of the entire Hex open reading frame allows one to examine Hex function without disrupting potential protein-protein interactions. Expression of Hex-(lambda)VP2 in Xenopus inhibits expression of the anterior marker Cerberus and results in anterior truncations. Such embryos have multiple notochords and disorganised muscle tissue. These effects can occur in a cell non-autonomous manner, suggesting that one role of wild-type Hex is to specify anterior structures by suppressing signals that promote dorsal mesoderm formation. In support of this idea, over-expression of wild-type Hex causes cell non-autonomous dorso-anteriorization, as well as cell autonomous suppression of dorsal mesoderm. Suppression of dorsal mesoderm by Hex is accompanied by the down-regulation of Goosecoid and Chordin, while induction of dorsal mesoderm by Hex-(lambda)VP2 results in activation of these genes. Transient transfection experiments in ES cells suggest that Goosecoid is a direct target of Hex. Together, our results support a model in which Hex suppresses organiser activity and defines anterior identity.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 cer1 chrd gnao1 gsc hhex lgals4.2 nog
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Phenotypes: Xla Wt + dorsal hhex + Bgal (Fig. 4 BDEG) [+]

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	Fig. 1. Hex acts as a transcriptional repressor in ES cells. (A) GAL4- Hex represses transcription from upstream of the SV40 promoter. Increasing concentrations of DNA expressing either GAL4 or GAL4- Hex were co-transfected with the depicted reporter. (B) Cartoon illustrating the construction and principle behind the design of Hex- lVP fusions. (C) Activity of GAL4, GAL4-HexlVP2 and GALHex lVP4 in ES cells. DNA encoding the indicated fusion protein was co-transfected with the indicated reporters as in A. Bars represent mean activities in arbitrary units (see Materials and Methods); standard deviations are indicated. Amount of plasmids expressing the indicated GAL4 or Hex derivatives are indicated in A and is 100 ng for all transfections in C.
	Fig. 2. Phenotypes produced by Hex- lVP2 RNA injection. All injections used 25 pg Hex-lVP2 RNA and 100 pg nuclear b-galactosidase per blastomere. The schematic diagram in the lower right-hand corner indicates the stage, position and nature of each injection (red, Hex-lVP2 alone; yellow, b-galactosidase alone; orange, Hex-lVP2 and b-galactosidase; lime green, b-galactosidase, Hex-lVP2 and Hex). (A) Stage 36 control tadpole. (B,C) Hex-lVP2 RNA injected into two dorsal blastomeres at the 4-cell stage. (D) Hex-lVP2 RNA injected into two dorsal-vegetal blastomeres at the 8-cell stage. (E) b-galactosidase RNA injected into a single ventral-vegetal blastomere at the 8-cell stage. (F-H) b-galactosidase and Hex-lVP2 RNA injected into a single ventral-vegetal blastomere at the 8-cell stage. (I-J) b-galactosidase and Hex-lVP2 RNA injected into two dorsal blastomeres at the 4-cell stage. (K) b-galactosidase and Hex-lVP2 RNA injected two dorsal animal blastomeres at the 8-cell stage. (L) b-galactosidase, Hex-lVP2 and Hex RNA injected into two dorsal blastomeres at the 8-cell stage. (M,N) b- galactosidase and Hex-lVP2 RNA injected into two dorsal-vegetal blastomeres at the 8-cell stage. Frequencies for the phenotypes described above are listed in Table 1. For dorsal animal injections, 62% of cases were normal, 10% showed anterior truncations, 10% were cyclopic and 20% had non-specific gastrulation defects (n=21).
	Fig. 3. Hex-lVP2 induces dorsal mesoderm. Transverse sections through embryos described in Fig. 2. Dark blue stain is b-galactosidase lineage tracer and sections are stained with Feulgen, Light Green and Orange G. Muscle tissue and somite are denoted by an arrow, and notochord by an arrowhead. The type of injection is depicted in cartoon form in the upper right hand corner of each panel as in Fig. 2. (A) Control embryo at the hindbrain level, (B-E) dorsal injections, (F-G) dorsal-vegetal injection, (H) control embryo, (I-J) ventral-vegetal injection. The images in C, E, G and J are higher magnification views of sections in B, D, F and I.
	Fig. 4. Phenotypes produced by Hex RNA injection. Hex RNA (250 pg) was co-injected with a b-galactosidase lineage tracer into a single dorsal or a single dorsal-vegetal blastomere. (A) Control embryo injected with b-galactosidase lineage tracer. (B) Embryo injected with RNA encoding both Hex and b-galactosidase. (C,H) Sections through control embryos at comparable levels to D,E and I,J, respectively. Injection of Hex RNA caused either bulges around the mid-line (B) or loss of anterior and posterior axial structures (F,G). (D,E) Sections through the embryo in B showing disruption of the somite on the injected side (indicated by blue labelled cells), a yolky endoderm-like extension and induction/expansion of notochord. (I,J) Sections through the embryo in G showing a second neural tube in addition to disruption of the somite and induction of notochord on the injected side. Identification of notochord is confirmed by examination of serial sections. Antibody staining with the notochordspecific MZ15 shows these effects to be non-cell autonomous. (K) Cleared embryo injected with RNA encoding b-galactosidase, and stained with MZ15. (I) Embryo stained solely with MZ15 (orange); comparison with K reveals co-localisation of b- galactosidase and MZ15 staining. (M) Embryo injected with RNA encoding Hex has an enlarged notochord (orange) distinct from the population of Hex-injected cells (blue). (N) Embryo injected with Hex-lVP2 and stained with MZ15. The enlarged notochord shown in Fig. 3 is obscured by labeled cells which occupy axial positions. S, somite; NT, neural tube; NOT, notochord. After 4-cell dorsal injections (n=32), 33% showed a severe phenotype (Fig. 4F) and 66% a mild phenotype (Fig. 4B). After 8-cell dorsal injections (n=32), 19% showed a severe phenotype (Fig. 4F) and 81% a mild phenotype (Fig. 4B).
	Fig. 5. Injection of Hex-lVP2 RNA inhibits Cerberus expression. (A) Lateral view and (B) animal view of early gastrula stage 10-10.5 uninjected embryo. (C) Lateral view and (D) animal view of embryo injected with Hex-lVP2 RNA. White arrows indicates the deep expression domain of Cerberus and the black arrow the suprablastoporal domain. Embryos were injected with 25 pg of Hex- lVP2 RNA into both dorsal blastomeres at the 4-cell stage. All embryos were cleared in Murrayâs agent to visualize deep tissues.
	Fig. 6. Induction of dorsal mesoderm by Hex-lVP2 and suppression by Hex. (A) Injections of RNA encoding Hex-lVP2 induces ectopic Chd, causes down-regulation of BMP4 and has no effect on Noggin. (B) Injections of RNA encoding Hex inhibit expression of Chd and not Noggin. Embryos were co-injected with synthetic RNA and a fluorescent dextran lineage trace. In situ hybridization for the indicated marker is in blue and the lineage tracer is stained in red. All embryos were fixed at stage 10-10.5 and are orientated with dorsal upwards. The majority of injections were done at the 4- or 8-cell stage into ventral lateral blastomeres or in one case, at the top of (A), throughout the dorsal side (second panel from left). In B all embryos were injected at the 4-cell stage into either a single dorsal or ventral blastomere. Embryos were injected with either 25 pg of Hex-lVP2 RNA or 250 pg Hex RNA.
	Fig. 7. Regulation of Goosecoid by Hex. (A) Injection of Hex-lVP2 RNA induces ectopic Gsc expression. Gsc expression is in blue and the fluorescent dextran lineage tracer is in red. (B) Injection of Hex RNA leads to a reduction in Gsc expression. Equivalent concentrations of Hex or b-galactosidase RNA (1 ng), as a negative control, were injected into embryos at the 2-cell stage, on the dorsal side at 4-cell stage or, in the bottom panel, 500 pg of Hex RNA was injected into either a single dorsal or a single ventral blastomere at the 4-cell stage (the arrow indicates the location of the injection relative to the endogenous Gsc domain). As in (A), in situ hybridization for Gsc is in blue and the lineage tracer is red. The stage and orientation of embryos is as Fig. 6. (C) Hex and Hex-lVP2 activity in animal cap explants. Hex RNA and EF-1a, as an internal control, were detected by RT-PCR. The bar over the second set of lanes indicates that the animal cap explants were cultured in the presence of 8 units/ml of activin, to induce Gsc. Embryos were injected with the same amount of RNA as those in Fig. 5 unless otherwise indicated.
	Fig. 8. Hex recognises the Goosecoid promoter in ES cells. (A) Deletion analysis of the activin response element in the Gsc promoter by transient transfection with DNA encoding Hex-lVP2. (B) Activity of Hex- lVP2, GAL4-Hex-lVP2 and different point mutations in Hex from the Gsc promoter. (C) Activity of GAL4- Hex-lVP2 and point mutations from 5 GAL4 binding sites. The level of activation of Gsc is fold induction. The data represented here is based on multiple experiments in which the basal levels have all been normalized to 1. Error bars represent standard deviations. The overall activity in C is the total level of transcription from this promoter. Each number represents an average of multiple independent transfections with the error bar representing the standard deviation. Arrows indicate transfection of increasing amounts of DNA encoding the protein indicated. In A and B 200, 400 or 800 ng of a plasmid expressing the indicated Hex derivative were cotransfected with 100 ng of the indicated reporter. In C a range of concentrations of GAL4-Hex-lVP was transfected (100-600 ng), while subsets of these values (one low point and one high point) were used for all the other indicated Hex derivatives.
	Fig. 9. Hex-lVP2 but not Hex(N186P)-lVP2, or Hex(N186K)-lVP2 induces Gsc. All injections were 25 pg of RNA in a ventral position at the 16-cell stage.
	Fig. 10. Spatial expression patterns of Gsc and Hex during gastrulation. In situ hybridization in near-adjacent sections of Xenopus embryos with probes to Hex and Gsc. A,C,E and G are stained for Hex and B,D,F,H are stained for Gsc. Embryo in A and B is stage 9, embryo in C and D is stage 10-10.5, embryo in E and F is stage 10.5-11, embryo in G and H is stage 11-11.5. The sections in G and H go through the dorsal side at an angle that is imperfectly transverse. Thus anterior is at the top and dorsal extends down to the right.