ETAPAS DE UN ESTUDIO DE SIMULACIÓN

It is thought that the Hox genes themselves represent a molecular code that specifies positional information along the AP axis during development. This is often referred to as the

Hox code (Kessel & Gruss, 1991; Hunt & Krumlauf, 1992; McGinnis & Krumlauf, 1992; Krumlauf, 1994). It is well established that Drosophila HOM-C genes specify positional information during embryogenesis and their extensive similarity to the vertebrate Hox genes suggests that they have similar functions. Direct evidence for the functional significance of the Hox code, indicating that the precise setting of the boundaries of Hox gene expression is crucial for orderly development, has been derived from; loss- and gain-of-function mutational analyses of the Hox genes which produce characteristic homeotic transformation phenotypes (see Chapter 1.2.6), in vivo modulation of Hox genes by RA (Kessel & Gruss, 1991; Kessel, 1992, 1993; Marshall et a l, 1992; Wood et a l, 1994), and transplantation experiments (Guthrie e ta l, 1992).

Despite extensive characterisation of the Hox/HOM complexes the underlying requirement for their striking structural conservation throughout evolution, and the relationship of this to colinearity, remains a mystery. It seems most likely, however, that this has been imposed by functional constraints. It has been postulated that the integrity of the entire Hox cluster is required for the proper regulation and expression of the genes within it. This can be viewed from two extreme standpoints. Firstly, within the Hox complex there exists a single 'master control region', located towards the 3' end of the cluster. This region would be critical for governing the characteristic colinear patterns of Hox gene expression that are observed. A similar type of regulatory mechanism has been well characterised for the expression of the developmentally regulated human 6-globin gene cluster (Grosveld et a l, 1987; Dillon & Gros veld, 1993). In this case, a combination of stage-specific factors acting at proximal cis-

regulatory sequences, and the competition of individual promoters for long-range interaction with the Locus Control Region (LCR), ensures correct sequential developmental gene regulation. The physical interactions between the LCR and individual promoters is thought to occur by a DNA-looping mechanism. The contrasting scenario is that multiple enhancer elements, scattered throughout the length of the Hox cluster, are required for the appropriate regulation of one or several genes. In this type of model the dispersed regulatory elements

would act like 'links of a chain', holding the Hox cluster together. It is possible that the genuine mechanism underlying the coordinated expression of H ox/H O M genes lies somewhere between these two extreme perspectives, involving both long-range and local interactions of c/j-regulatory elements acting on single or multiple transcriptional units. Some supporting evidence for this theory may be derived from the results of numerous experiments aimed at recreating the full expression patterns of individual Hox genes in transgenic mice. Employing reporter gene analysis it has been possible to recapitulate the endogenous pattern of gene expression in a few instances, using relatively small genomic regions (approximately 6-18kb), out of their natural genomic context, i.e. for H o xa -7

(Piischel et al., 1991), Hoxb-4 (Whiting et a l, 1991), Hoxb-1 (Marshall et al., 1992) and

Hoxa-4 (Behringer et a l, 1993). It must be stressed that such reporter gene assays are only valid for the assessment of the crudest aspects of gene expression such as the spatial and, to a lesser extent, temporal distribution of transcripts. As two of the desired properties of a reporter gene assay are heightened sensitivity and stability, no conclusions may be inferred about the precise levels of gene expression nor about the nuances of temporal variation. These results are, however, in accordance with the observation that all of the sequences of the the Drosophila BX-C do not need to be contiguous in order to function properly (Struhl, 1984; Tiong et a l, 1987). This tends to argue against a straightforward LCR-based model of

Hox gene regulation.

In all of the cases where endogenous Hox gene expression has been faithfully reproduced, reporter constructs include genomic sequences located both 5' and 3' of the transcription start site. It is clear from these limited examples that the mechanisms employed in H ox gene transcriptional regulation are diverse. For example, for both Hoxa-7 and Hoxb-4 multiple regulatory elements located downstream of the transcription start site are required, but have contrasting modes of action. In the case of Hoxa-7, the promoter (5'-flanking DNA) is active in all regions of the embryo and the regulatory elements act to restrict this activity. In Hoxb- 4 the promoter is essentially inactive and appropriate expression is only seen in the presence of the regulatory elements. It is also interesting to note that the paralogous genes Hoxa-4 and

Hoxb-4 do not share common spatial arrangements of regulatory sequences, since in Hoxa-4

the major regulatory elements appear to be located upstream of the promoter rather than downstream. Hoxa-7 and Hoxb-7 (Vogels et a l, 1993) are also dissimilar in two respects. Firstly the regulation of H oxb-7 is predominantly dependent on activating upstream cis-

regulatory sequences, rather than on upstream and downstream lineage-restricting elements. Secondly, 27kb of genomic sequences encompassing the Hoxb-7 gene are unable to recapitulate the full expression pattern of the endogenous gene, whereas for H oxa -7

approximately 7kb was sufficient. In the majority of instances endogenous Hox gene expression patterns have only been partially reproduced with varying degrees of success, for example; Hoxa-1 and Hoxa-2 (Frasch et a l, 1995), Hoxa-5 (Zakany et a l, 1988; Tuggle et

a l , 1990), Hoxb-6 (Schughart et a l, 1991; Eid et a l, 1993); Hoxb-7 (Vogels et a l, 1993),

Hoxc-8 (Bieberich et a l, 1990; Shashikant et a l, 1995), and Hoxd-11 (Gérard et a l, 1993). Admittedly some of these analyses have only focused on upstream sequences. However, the inability to recreate the endogenous expression pattern of some of the Hox genes suggests that dispersed, and possibly shared, regulatory elements are required. Crucially this line of research has uncovered numerous cw-regulatory elements that are important for particular aspects of Hox gene expression, some of which are capable of acting as spatially-specific enhancers (e.g. Whiting et a l, 1991). Thus the identification of fm»j-acting factors that interact with these sequences will provide important information about how Hox gene expression is activated and maintained in vertebrates.

The presence of interwoven regulatory elements may explain how the Hox/HOM genes have remained clustered but does not explain why this is necessary for ordered, temporally and spatially colinear Hox gene activation. In considering this conundrum we may not be as justified in turning to the Drosophila HOM -C for a comparison with the vertebrate Hox

Complex as we have been in other instances. As discussed earlier in this text, many fundamental differences exist between Drosophila and murine embryogenesis at both the molecular and cellular level, particularly with respect to pattern formation. Most evident is the presence of a molecular 'pre-patteming' mechanism involving matemal-effect, gap, pair- rule and segment-polarity genes that predisposes the expression of the HOM-C genes. There is no evidence to suggest that such a system operates in vertebrates, although later phases of

Hox/HOM expression appear to utilise analogous mechanisms for the maintenance of the 'on/off state (Krumlauf, 1994). Some of the genes involved in H O M -C regulation are conserved in vertebrates, and in certain cases are known to be involved in aspects of Hox

gene regulation (see Chapter 1.4). However, this does not necessarily mean a directly conserved role in development per se (Lobe & Gruss, 1989). It is possible that the evolution of a pre-patteming system in Drosophila has diverted some degree of evolutionary pressure away from the organisational requirement of HOM-C, particularly in terms of the relative mechanisms for the initial activation of Hox/HOM genes (Kenyon, 1994). This would help to explain some of the more divergent characteristics associated with HOM-C, when compared to Hox clusters on the chordate/vertebrate lineage (Kmmlauf, 1994). For example, the split nature of HOM-C and the presence of other non-Hox/HOM like genes that are dispersed amid

ANT-C.

Despite the possible differences in the initial mechanisms of activation of Hox/HOM genes, temporal and spatial colinear expression has been described in vertebrates, short germ-band insects such as the locust Schistocerca, and leeches (Kmmlauf, 1992, 1994; Akam, 1994; Shankland, 1994). This process is not dependent on spatial cues as it has been observed in cell culture in response to RA treatment, as previously noted. Thus, the key to the conserved

clustered genomic arrangement of Hox genes may be to permit ordered temporal expression, resulting in ordered spatial expression. Gaunt & Singh (1990) have suggested that a bipartite mechanism of transcriptional activation and maintenance could explain the features of temporally and spatially colinear expression of the Hox gene complexes. During activation a progressive 3' to 5' opening up of a cluster (i.e. in the same order that the genes are expressed along the AP axis) would result from the activity of stage- and spatially-restricted morphogens on the c/5-regulatory elements of individual genes. Subsequently the active/inactive state of each gene would be heritably maintained by a silencing mechanism, e.g. by the recruitment of Pc-G-like proteins as previously described. The requirement for gene clustering would therefore be at the level of the maintenance of transcriptional repression. Duboule (1992) proposed a variation on this model which perhaps seems more logical as it places the emphasis for the clustered arrangement at the level of transcriptional activation. This model states; an imperative reason for the conserved clustering of the Hox

genes might be the need for a carefully controlled sequential activation of the genes from 3' to 5' in the clusters during embryogenesis. Hox gene clustering would be required for the fine tuning of the temporal sequence in which the genes are activated (by a still unknown molecular mechanism) during the transmission of positional information in embryos. For this model to hold true, the 'unknown molecular mechanism' would still need to stimulate Hox

gene activation in a temporal manner, such that the more posterior cells in the embryo activate their Hox gene expression later than more anterior ones. In truth of fact, only when we have a gained a greater understanding of Hox gene expression will we be able to resolve the mechanisms by which colinearity is achieved. Hence the dissection of the regulatory mechanisms governing Hox gene regulation is the focus of much research.