Ladher R et al. (1996), Xom: a Xenopus homeobox gene that mediates the ...

XB-ART-17921

Development 1996 Aug 01;1228:2385-94. doi: 10.1242/dev.122.8.2385.

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Xom: a Xenopus homeobox gene that mediates the early effects of BMP-4.

Ladher R , Mohun TJ , Smith JC , Snape AM .

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Bone morphogenetic protein-4 (BMP-4) is thought to play an important role in early Xenopus development by acting as a "ventralizing factor' and as an epidermal determinant: local inhibition of BMP-4 function in whole embryos causes the formation of an additional dorsal axis, and inhibition of BMP-4 function in isolated ectodermal cells causes the formation of neural tissue. In this paper we describe a homeobox-containing gene whose expression pattern is similar to that of BMP-4, whose expression requires BMP-4 signalling and which, when over-expressed, causes a phenotype similar to that caused by over-expression of BMP-4. We suggest that this gene, which we call Xom, acts downstream of BMP-4 to mediate its effects.

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Species referenced: Xenopus
Genes referenced: actc1 actl6a bmp2 bmp4 fgf2 hba4 nog odc1 tbxt ventx2.2

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	Fig. 1. (A) Xom cDNA sequence and deduced amino acid sequence. Amino acids in bold represent the homeodomain. The threonine at position 47 in this homeodomain is underlined, as is the polyadenylation signal at nucleotides 1226-1231. (B) Comparison of the homeodomain of Xom with those of members of the same homeodomain class. The threonine at position 47 is again underlined. (C) Structure of Xom. The acidic domain (amino acids 41 to 172; red) is defined as a region in which the average charge over 20 amino acids is consistently negative. In the middle of this domain is a region rich in serine and threonine (diagonal stripes). The homeodomain is contained within amino acids 173-233 (blue). The proline-rich domain (amino acids 255-320) contains 14 proline residues in a stretch of 66 amino acids (yellow).
	Fig. 2. RNAase protection analysis of Xom expression. Xom transcripts are first detectable at stage 9 and expression declines after stage 17. Ornithine decarboxylase (ODC) is used as a loading control.
	Fig. 3. Whole-mount in situ analysis of Xom expression. (A) Vegetal view of a Xenopus embryo at stage 9 shows asymmetric expression of Xom. It is likely that Xom is most highly expressed in ventral cells, because by stage 10.5 transcripts are clearly excluded from the organizer (B). Apparent lack of expression in vegetal tissue is probably due to poor probe penetration. (C) Dorsal view of an embryo at stage 12.5; expression is excluded from the anterior neural plate and the dorsal midline. (D) Dorsal view of an embryo at stage 14. Expression is becoming restricted to two domains, one anterior and one posterior. The posterior domain surrounds the lateral and ventral regions of the embryo. (E) Lateral view of an embryo at stage 20. The anterior domain has resolved to mark the dorsal region of the eye. The posterior domain includes the ventral region of the embryo and (out of the plane of the photograph) the dorsal midline in the trunk. (F) Lateral view of an embryo at stage 32 shows expression of Xom in the dorsal part of the eye, in the proctodeum and in the tailbud. Scattered expression is also visible in the dorsal midline, which may be due to migrating neural crest cells.
	Fig. 4. Sections of embryos processed for whole-mount in situ hybridization using a probe specific for Xom. (A) Section of stage- 10.5 embryo. Transcripts are absent from the organizer, but are present in ventral and lateral regions of the embryo in all three germ layers. The punctate staining in vegetal tissue may be due to poor probe penetration into this yolky tissue. (B) Section of a stage-19 embryo at the level of the head. Transcripts are present in the presumptive eye. (C) Section of the embryo shown in B in the trunk region. Staining is visible in all three germ layers of the ventral and lateral regions of the embryo, and in the dorsal cells of the roof plate of the neural tube. Bars, in A and B are 50 mm; in C is 100 mm.
	Fig. 5. Expression of Xom is prevented by dispersion of Xenopus embryos. Addition of activin or BMP-4, but not FGF, restores expression. Xenopus embryos were transferred to calcium- and magnesium-free medium at stage 7.5, their vitelline membranes were removed, and the blastomeres were kept dispersed by passing a gentle stream of medium over the cells from a Pasteur pipette. Dispersed blastomeres were cultured to stage 10.5 in the presence of the indicated factors and analysed for expression of Xom by RNAase protection. EF-1a was used as a loading control. Lane 1, intact control embryos; lane 2, dispersed blastomeres with no additional factors; lane 3, dispersed blastomeres plus 50 ng/ml FGF-2; lane 4, dispersed blastomeres plus 100 ng/ml BMP-4; lane 5, dispersed blastomeres plus 8 units/ml activin.
	Fig. 6. Activation of Xom expression by activin and BMP-4 is not inhibited by cycloheximide. Xenopus embryos were transferred to calcium- and magnesium-free medium at stage 7.5, their vitelline membranes were removed, and the blastomeres were kept dispersed by passing a gentle stream of medium over the cells from a Pasteur pipette. Dispersed blastomeres were cultured to stage 10.5 in the absence of additional factors (lanes 1 and 2) or in the presence of FGF-2 (lanes 3 and 4), BMP-4 (lanes 5 and 6) or activin (lanes 7 and 8). Samples in even-numbered lanes were cultured in the continual presence of 7.5 mg ml-1 cycloheximide. This was sufficient to reduce incorporation of [35S]methionine into trichloroacetic acid-insoluble material by over 94%. Expression of Xom was analysed by RNAase protection. EF-1a was used as a loading control.
	Fig. 7. Injection of RNA encoding noggin into one blastomere of Xenopus embryos at the 2-cell stage inhibits expression of Xom. Noggin RNA (1 ng) was injected into one blastomere of Xenopus embryos at the 2-cell stage and the embryos were allowed to develop to the early gastrula stage (stage 10), when they were analysed by whole-mount in situ hybridization. (A) Noggininjected embryo viewed from the animal hemisphere. Note downregulation of Xom expression in half the embryo (arrow). (B) Control embryo viewed from the animal hemisphere.
	Fig. 8. Expression of a truncated BMP-4 receptor blocks early expression of Xom, and over-expression of BMP-4 causes ectopic expression. (A) RNA encoding a truncated BMP-2/4 receptor (tBR) was injected into Xenopus embryos at the 1-cell stage and the embryos were allowed to develop to the indicated stages before being analysed by RNAase protection using a probe specific for Xom. At stages 8 and 9 there is a dramatic down-regulation of Xom compared to uninjected controls, but this has recovered by stage 11. EF- 1a was used as a loading control. (B) In situ hybridization analysis of embryos injected with a truncated BMP-2/4 at stage 10 also reveals inhibition of Xom expression. (C) Overexpression of BMP-4 causes expression of Xom to occur in the dorsal marginal zone as well as in ventral tissues.
	Fig. 9. Comparison of the time of onset of expression of Xom and of BMP-4. Xenopus embryos at the indicated times after fertilisation (hours) were analysed by RNAase protection simultaneously for the expression of BMP-4, Xom and EF-1a. Low maternal levels of BMP-4 RNA are visible and zygotic expression of Xom slightly precedes that of BMP-4.
	Fig. 10. Injection of RNA encoding Xom into dorsal blastomeres of Xenopus embryos causes loss of anterior structures and of the notochord. Xom RNA (4 ng total) was injected into the two dorsal blastomeres of Xenopus embryos at the 4-cell stage, and the embryos were allowed to develop to stage 32. Control embryos received injections of RNA encoding DXom, which encodes a truncated version of Xom. (A) Embryos expressing DXom develop normally. (B) Embryos injected with RNA encoding Xom lack anterior structures. (C) Whole-mount antibody staining using the monoclonal antibody MZ15 demonstrates that embryos injected with DXom RNA form a notochord. (D) Embryos injected with RNA encoding Xom lack a notochord.
	Fig. 11. Xom, but not DXom, changes the fate of prospective dorsal mesodermal cells. RNA (2 ng) encoding DXom (A, B), or Xom (C, D) was injected into blastomere C1 of Xenopus embryos at the 32-cell stage along with RNA (100 pg) encoding b- galactosidase, which acts as a lineage marker. The embryos were allowed to develop to stage 42, when they were fixed and processed for b-galactosidase expression. Note that blastomeres injected with RNA encoding DXom make a major contribution to notochord (A, B), while those expressing Xom (C, D) preferentially populate the somites.
	Fig. 12. Animal caps derived from embryos injected with Xom RNA do not elongate in response to activin. Animal caps were dissected from control embryos or from embryos injected with RNA encoding Xom or DXom. They were left untreated or were exposed to 4 units/ml activin. Caps were photographed at the equivalent of stage 17. (A) Control animal caps remain spherical. (B) Activin treatment of animal caps derived from uninjected embryos causes dramatic elongation. (C) Animal caps derived from embryos injected with RNA encoding DXom remain spherical. (D) Activin treatment of animal caps derived from embryos injected with RNA encoding DXom causes elongation. (E) Animal caps derived from embryos injected with RNA encoding Xom remain spherical. (F) Activin treatment of animal caps derived from embryos injected with RNA encoding Xom does not cause elongation.
	Fig. 13. Xom inhibits induction of dorsal mesoderm by activin but does not affect activation of Xbra or induce expression of aT4 globin. Animal caps derived from embryos injected with RNA encoding Xom or DXom were exposed, where appropriate, to activin (4 units/ml) and cultured to the equivalent of stage 25 for analysis of cardiac actin expression, to stage 10.5 for analysis of Xbra and to stage 25 for analysis of aT4 globin. (A) Induction of cardiac (muscle-specific) actin is inhibited by Xom RNA but not by DXom. (B) Expression of Xbra is not affected by Xom and Xom does not induce expression of aT4 globin, even though activin-induced elongation in this experiment was blocked (not shown). Expression of EF-1a was used as a loading control.