XB-ART-4826Development October 1, 2003; 130 (19): 4611-22.
Members of the Bix family of homeobox-containing genes are expressed in the vegetal hemisphere of the Xenopus embryo at the early gastrula stage. Misexpression of at least some of the family members causes activation of mesoderm- and endoderm-specific genes and it is known that some of the proteins, including Bix2 and Bix3, interact with Smad proteins via a motif that is also present in the related protein Mixer. In this paper we study the function of Bix3. Misexpression of Bix3, similar to misexpression of other members of the Bix family, causes the activation of a range of mesendodermal genes, but the spectrum of genes induced by Bix3 differs from that induced by Bix1. More significantly, we find that overexpression of Bix3 also causes apoptosis, as does depletion of Bix3 by use of antisense morpholino oligonucleotides. The ability of Bix3 to causes apoptosis is not associated with its ability to activate transcription and nor with its possession of a Smad interaction motif. Rather, Bix3 lacks a C-terminal motif, which, in Bix1, acts in cis to inhibit apoptosis. Mutation of this sequence in Bix1 causes the protein to acquire apoptosis-inducing activity.
PubMed ID: 12925588
Article link: Development
Genes referenced: bcl2 bix1.1 bix1.2 bix1.3 bix2 bix3 mixer
Morpholinos referenced: bix3 MO1
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|Fig. 1. The expression patterns of Bix1 and Bix3. In situ hybridisation was performed on bisected embryos at the indicated stages using probes specific for Bix1 and Bix3. In C-H, dorsal is to the right.|
|Fig. 4. Apoptosis in Xenopus embryos injected with RNA encoding Bix3. (A-F) One group of embryos was left uninjected whereas another (G-I) was injected with 400 pg of Bix3 RNA at the one-cell stage. At stage 8.5, half of the uninjected embryos were transferred to 0.1 mg/ml cycloheximide (D-F), whereas the other half was allowed to develop normally (A-C). Embryos were collected at the indicated stages and used for TUNEL staining. Positive cells are dark blue. Note that loosely adherent cells in Bix3-injected embryos, as seen in Fig. 2, are lost during the TUNEL procedure. (J) tPARP assay shows that Bix1 does not activate caspase activity whereas induction of apoptosis by Bix3 was first detectable by stage 11. Cycloheximide induces caspase activity from early gastrula stage 10. Intact tPARP has a relative molecular mass of 60 kD (upper arrows in both gels), and this is cleaved into fragments of 36 kD and 24 kD (lower arrows). (K) Human Bcl2 delays the onset of Bix3-induced apoptosis. Caspase assays were performed on control embryos at the indicated stages, embryos injected with RNA encoding Bix3, or embryos injected with Bix3 RNA together with RNA encoding human Bcl2 (500 pg). Note that human Bcl2 delays the onset of caspase activity. (L-N) Partial rescue of the cell adhesion and apoptosis defect by human Bcl2. Bix3 RNA alone (50 pg; L), or in combination with 500 pg of human Bcl2 RNA (M), was injected in the animal pole of one-cell stage embryos. Embryos were left to develop and photographed at mid-gastrula stage 11.5-12. Note that human Bcl2 does not prevent the appearance of darkly pigmented cells in the animal hemisphere but does reduce or delay the incidence of cell disaggregation. Human Bcl2 delayed disaggregation in five out of seven experiments of this sort. (N) Quantitation of an experiment of the type illustrated in (L) and (M). Embryos undergoing cell disaggregation were scored at the indicated stages.|