MARCO CONCEPTUAL

In this thesis I have described the cloning of Xom, based on its homology with the homeobox-containing gene family. Xom contains a homeodomain and is therefore postulated to act as a DNA binding protein. The homeodomain of Xom is of an unusual class, marked by the presence of a threonine in position 47 of the homeodomain, in place of the more usual valine or isoleucine. In another member of this class this residue has been shown to be important in determining the DNA binding specificity of the

homeodomain. Hox 11, a. gene required for the development of the mouse spleen (Dear et a l , 1995), also contains a threonine at position 47 of the homeodomain. In H ox 11 this threonine causes the binding specificity of the homeodomain to change from 5 ’-TAAT-3’ to the more unusual 5 -TAAC-3' (Dear et a l, 1993). Similarly, the DNA binding activity of another member of this class. Bar H I, is also different from most other homeodomains (Kalionis and O'Farrell, 1993). It should be noted that the homeodomain of B arH l is

most related to the homeodomain of Xom. It is therefore possible that the DNA binding specificity of Xom is also changed. It is also interesting that the homeodomains o f other ventralising Xenopus homeobox genes have the threonine substitution at position 47 (Gawantka et a l, 1995; Schmidt et a l, 1996). It is unclear why the embryo has so many homeobox genes with a similar expression pattern, similar activity and with a threonine at position 47 of the homeodomain. Perhaps these genes act as a complex to bind a DNA. This may be why Xom has a rather weak ventralising activity. A prediction from these obsevations is that downstream genes of the ventralising homeobox genes will all contain a similar promoter sequence, possibly containing the TA AC motif.

8.2. The expression of Xom

Xom is expressed very early, probably with the onset of zygotic transcription. It is

important therefore to compare the expression of Xom with other genes that are expressed around this time, such as goosecoid and Xwnt-S, in order to place these genes into a potential regulatory hierarchy. It is known, for example, that the gene siamois is

expressed before goosecoid and Xwnt-8 (Lemaire et a l, 1995). RNAase protection data similar to that performed for Xom and BMP-4 (figure 6.8) would indicate position of

Xom in this chain.

In a similar fashion, the onset of transcription of the ventralising homeobox genes could be compared to reveal some clues to their function and to determine if a similar hierarchy exists amongst them. As yet, data on the onset of the transcription o f Vox

(Schmidt et a l, 1996) and pv-1 (M. Jamrich; personal communication) is unavailable, however reports that Xvent expression only commences at gastmla stages (Gawantka et a l , 1995), places its transcripton after that of Xom.

8.3. The induction of Xom

From the data presented in this thesis, the action of BMP-4 is central to the expression of

perturbations in BMP-4 signalling also perturb the expression pattern of Xom. This is of interest as the suggestion that BMP-4 performs an inductive function in the embryo is supported by a strong body of evidence. BMP-4 is expressed in the correct place at the correct time for its proposed ventralising role (Schmidt et a l, 1995). Its effects on

isolated tissue also have the postulated effects (Dale et a l, 1992; Jones et al., 1996; Jones

et al., 1992). Inhibition of the BMP pathway using either a truncated BMP receptor (for example see (Suzuki et a l, 1994)) or by using "natural" inhibitors such as noggin (Smith and Harland, 1992; Smith et a l, 1993) or chordin (Holley et ah, 1995; Sasai et a l, 1994) inhibit the formation of ventral tissue, with a compensatory increase in dorsal structures.

Xom expression is localised as soon as it is detectable by whole mount in situ hybridisation, and this localisation probably marks the ventral side of the embryo. Data using the truncated BMP receptor suggests that this expression is controlled by maternal BM P signalling. However it is unclear whether maternal levels of BMP-2 or BMP-4 are responsible for this induction. Experiments using antisense oligonucleotides to prevent translation from a maternal RNA such as BMP-2 or 4 may go some way to addressing the exact identity of the inducing molecule (see, for example, (Sasai et a l, 1995)), but would still leave the conundrum of how Xom is localised in the first place. It is possible that a maternal BMP is able to act throughout the margin of the embryo but is prevented from doing so on the dorsal side by the action of localised maternal noggin. Again experiments studying the change in Xom localisation when antisense oligonucleotides prevent

maternal noggin translation should address this. Another possibility is that cortical rotation not only establishes the dorsal side of the embryo; it also molecularly defines the ventral side. If this is true, Xom should be expressed throughout the embryo, at pre­ gas trulation stages of development, when cortical rotation is inhibited by ultra-violet irradiation of the vegetal pole.

The expression pattern of Xom seems to be almost identical with that o f BMP-4. This, together with the observation that BMP-4 can induce Xom in an immediate early

fashion, strengthens the argument that Xom is a transcriptional response to BMP-4 activity. However there are regions where the later expression of the two genes do not overlap. BMP-4 is in the heart region of the embryo and Xom is not; Xom is in the tailbud and BMP-4 is not. The tailbud expression is intriguing, suggesting that another mechanism, not dependent on BMP-4 signalling, is responsible for expression here. Alternatively, the levels of BMP-4 in the tailbud may be too low to be detected by the whole mount in situ hybridisation procedure.

8.4. The Function of Xom

The presence of a homeobox in the sequence of Xom suggests that it can act as a

transcription factor. An acidic domain N-terminal to the homeodomain and a proline-rich domain on the C-terminal side suggest that Xom is able to act as a transcriptional

activator (Ma and Ptashne, 1987; Mermod et a i, 1989). The identity o f target genes of

Xom is unknown, and it would be of interest to screen for possible downstream responses o f Xom.

Further clues on the function of Xom may be gained from the using a "dominant negative" approach to interfere the activity of Xom. One possible method that can be used is a procedure developed for the homeobox gene, Mix-1 (P. Mead and L. Zon;

unpublished observations). Here an interfering allele of Mix-1 was constructed by mutating a residue in the homeodomain. This allele bocks the activity of Mix-1. The effects of a similar construct made for Xom could be compared to the effects o f the dominant negative BMP receptor to provide more information on the activity of Xom.

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