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, 28 (17), 1879-84

Pax6-dependent, but β-Catenin-Independent, Function of Bcl9 Proteins in Mouse Lens Development

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Pax6-dependent, but β-Catenin-Independent, Function of Bcl9 Proteins in Mouse Lens Development

Claudio Cantù et al. Genes Dev.

Abstract

Bcl9 and Bcl9l (Bcl9/9l) encode Wnt signaling components that mediate the interaction between β-catenin and Pygopus (Pygo) via two evolutionarily conserved domains, HD1 and HD2, respectively. We generated mouse strains lacking these domains to probe the β-catenin-dependent and β-catenin-independent roles of Bcl9/9l and Pygo during mouse development. While lens development is critically dependent on the presence of the HD1 domain, it is not affected by the lack of the HD2 domain, indicating that Bcl9/9l act in this context in a β-catenin-independent manner. Furthermore, we uncover a new regulatory circuit in which Pax6, the master regulator of eye development, directly activates Bcl9/9l transcription.

Keywords: Bcl9; Pax6; Pygopus; Wnt; lens induction; mouse.

Figures

Figure 1.
Figure 1.
The Bcl9/9l–Pygo2 interaction is necessary during mouse embryonic development. (A) Wild-type and ΔHD1 mutant Bcl9 and Bcl9l proteins; at the right is presented the variation in the “chain of adaptors” induced by the deletion of the HD1 domain. (WRE) Wnt-responsive element; (PHD) plant homeodomain; (NHD) N-terminal homology domain. (B) A GST-Bcl9-ΔHD1 protein, when incubated with protein extracts obtained from 12.5-dpc wild-type embryos, loses the ability to interact with Pygo2 but maintains the ability to pull down β-catenin; nonspecific signal (n.s.), obtained with Pygo2 antibody, was used as a loading control. (C) Crossings between double-heterozygous mice: All of the possible genetic combinations are grouped based on the number of wild-type alleles of Bcl9/9l, from four out of four to zero out of four. Double-homozygous ΔHD1 mutant mice (Bcl9/9l-ΔHD1) are never found after 13.5 dpc, indicating embryonic lethality. (D) Bcl9/9l-ΔHD1 mice at 13.5 dpc appear slightly smaller but do not display any obvious developmental defect, apart from an eye malformation (see Fig. 2), when compared with control littermates (wild type [WT]).
Figure 2.
Figure 2.
Bcl9/9l has a role in early lens development. (Top panels) Bcl9/9l-ΔHD1 double-mutant embryos display an eye defect highly reminiscent of the one previously described for Pygo2 loss of function (Song et al. 2007). (Middle panels) The dissection of the eye structure at this stage (i.e., 13.5 dpc) shows a complete absence of the lens accompanied by an enlarged developing retina, a feature that resembles the lens-specific conditional loss of Pax6 (Ashery-Padan 2000). (Bottom panels) Bcl9/9l-ΔHD1 mutant embryos at 10.5 dpc fail to induce eye placode thickening and subsequent lens pit formation. A square bracket indicates the lens vesicle in the wild-type (WT) eye and the region of the surface head ectoderm that, in Bcl9/9l-ΔHD1 mutants, despite lying close to the optic vesicle, fails to form a lens placode.
Figure 3.
Figure 3.
Bcl9/9l act independently from β-catenin during lens development. (A) Wild-type (WT) and ΔHD2 mutant Bcl9 and Bcl9l proteins; at the right is presented the variation in the “chain of adaptors” induced by deleting the HD2 domain: The interaction between Bcl9/9l and β-catenin is abrogated, and the complex Bcl9/9l–Pygo2 can act independently from canonical Wnt signaling. (B) A GST-Bcl9-ΔHD2 protein loses the ability to interact with β-catenin but maintains the ability to pull down Pygo2. (C) No double-homozygous ΔHD2 mutant mice are ever scored after birth, suggesting embryonic lethality. (D) The few embryos found at 12.5 dpc displayed an evident developmental block at 10.5 dpc. (E) At 10.5 dpc, wild-type surface head ectoderm invaginates to form the lens pit; in Bcl9/9l-ΔHD1 embryos, this process is arrested (cf. the middle and left panels). At the same stage, Bcl9/9l-ΔHD2 embryos have a correctly shaped lens pit, demonstrating that, in this process, Bcl9/9l function is independent from β-catenin.
Figure 4.
Figure 4.
Bcl9 and Bcl9l are direct transcriptional targets of Pax6. (A) The mouse-derived lens cell line αTN4 expresses Bcl9, Bcl9l, and Pygo2 together with lens-specific genes such as Pax6 and Cryaa. (B) ChIP performed on the chromatin extracted from αTN4: Specific regions upstream of Bcl9 and Bcl9l are enriched when the chromatin is immunoprecipitated with an anti-Pax6 antibody, indicative of Pax6-binding events. No such enrichment was scored within the Pygo2 promoter. Cryaa and Prm2 promoters constitute the positive and the negative controls, respectively. The enrichment is expressed as a ratio between anti-Pax6 and control IgG immunoprecipitation reactions. (TTS) Transcriptional start site. (C) When αTN4 is treated with siRNA against Pax6, Pax6 mRNA is reduced to <5% of the control. As its direct target, Cryaa, Bcl9 and Bcl9l levels are also reduced upon Pax6 depletion. Of note, Pygo2 transcription is not altered. (D) A loss-of-function mutation of Pax6 in vivo leads to a diminished Bcl9l expression in the surface head ectoderm at the onset of lens pit formation. Arrows mark the surface head ectoderm (already forming the lens pit in the wild-type [WT] embryo); the asterisks indicate the diencephalic protrusion that constitutes the presumptive retina. DAPI is in blue and marks cell nuclei; Bcl9l is in red. (E) If αTN4 cells are treated with siRNA against Pygo2, Pax6 is down-regulated. In such experiments, Bcl9 and Bcl9l, together with Cryaa, are also affected, very likely due to a secondary effect of Pax6 down-regulation. (AU) Arbitrary units. (F, left panel) At 10.5 dpc, the surface head ectoderm that forms the lens pit expresses high levels of Pax6; at this stage in wild-type embryos, Pax6 appears to be stronger in the surface ectoderm (white arrows) than in the presumptive retina (asterisks). In Pygo2 knockout (Pygo2-KO) and Bcl9/9l-ΔHD1 embryos, this ratio is inverted. Please note that to detect the scant Pax6 within the surface head ectoderm, the signal must be enhanced; this explains the apparent brighter signal in mutant retinas. In Bcl9/9l-ΔHD2 embryos, the developing lens pit displays a Pax6 expression comparable with the wild type. Statistically significant values are evidenced by asterisks, which indicate a P-value < 0.05 calculated using an unpaired one-tail t-test.
Figure 5.
Figure 5.
Our data indicate that Pax6 is required for the transcription of Bcl9/9l. Bcl9/9l then assemble with Pygo2 to ensure a correct lens development; the lens development is arrested when the Bcl9/9l–Pygo2 interaction is abolished. The complex Bcl9/9l–Pygo2 also lies genetically upstream of Pax6, possibly with the function of sustaining its expression.

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