Expression of Hoxb-9 in the forelimb

At first glance, the forelimb pattem seen from constmct D appeared more similar to the

Hoxb-9 protein pattem than constmcts A to C. In both the antibody staining and constmct D there appears to be virtually no staining. In contrast, constmcts A, B and C, all gave expression in the posterior third of the forelimb. However, on more detailed examination, the protein pattem does contain a very small patch of expression in the posterior edge of the forelimb (Fig. 3.4i), and it may be the case that the transgenic expression seen in A to C, is a more sensitive reflection of this normal expression domain. Considereing that this same feature was seen in all three constmcts which contain sequences very close to the Hoxb-9 promoter, it is very unlikely that this strong, regular domain reflects an enhancer for a more distant gene. Furthermore, this pattem is also seen with the chicken Hoxb-9 gene, indicating that it is conserved (Pers. comm. L. McNaughton).

3.5 Summary

It has been shown that the majority of what is thought to be the normal expression pattem for the Hoxb-9 gene, can be controlled from an enhancer (or cluster of enhancers) which lies within the 2.9kb intron. This basic pattem appears to be complete for the mesodermal tissues, and in this respect is similar to Hoxb-4 which also contains a mesodermal enhancer within its intron. However, the late neural expression is not directed from this sequence, and it has been shown that the neural enhancer closest to the Hoxb-9 gene lies about 6kb 3 ’ from the promoter. Although in transgenic constmcts the anterior boundary of expression driven from this enhancer is not in exactly the correct position along the A-P axis, it is close enough to suggest that this is the important neural element for

Hoxb-9. There are no neural elements closer, or more similar in pattem to the Hoxb-9 gene, and the extra information required for its normal positioning may be derived from a global effect of die intact Hox complex. This 3 ’ positioning of a neural enhancer is also reflected in the Hoxb-4 and probably Hoxb-5 genes (see next chapter) which also possess a 3 ’ neural enhancer. It may therefore be an indication of the original gene duplication events which must have created the first Hox complex hundred’s of millions of years ago. This is discussed in more detail in the discussion.

CHAPTER 4

Regulatory interactions between adjacent Hox genes

The clustered organisation of the Hox genes, has led to two main questions: (1) Why do the genes need to be close to each other? (2) Does the proximity of promoters and enhancers from adjacent genes cause regulatory interactions to occur between the genes? In this part of the study I attempted experiments which relate to both of these questions.

Whiting etal. (Whiting et al., 1991), discovered that the main local regulatory elements for the Hoxb-4 gene, were located in three regions labelled A, B and C (Fig. 4.1). In transgenic experiments it was found that constmcts containing region A, plus the Hoxb-4 promoter and tiie LacZ gene, caused expression of P-galactosidase in the neural tube, from the posterior tip up to the boundary between rhombomeres 6 and 7 (r6/7). This is the same anterior boundary as the endogenous Hoxb-4 gene, and demonstrates that within region A is a spatially-specific neural enhancer for this gene. Similarly, in region C an enhancer was found which caused expression up to the correct anterior boundary in the somitic mesoderm (as well as some non-77ox6-4-specific neural expression). Expecting a similar set of enhancers to exist for the regulation of Hoxb-5, Stefan Nonchev in the lab, performed a series of transgenic experiments, in which different regions around the Hoxb-5 gene were tested. He defined two further regions, labelled D and B (Fig. 4.1), which appeared to be responsible for mesoderm and neural expression respectively. These regions are in the intergenic DNA between Hoxb-5 and Hoxb-4, and in further transgenic experiments it was shown that they could activate the Hoxb-4 promoter at least as well as the Hoxb-5 one. Thus arose the questions: Since these enhancers are near to both the Hoxb-4 and Hoxb-5 promoters, which one do they normally control? Could they be important to both? If they should only act on one promoter, how is interaction with the other one prevented?

The experiments described in this chapter were an attempt to prove whether any enhancers between adjacent genes can operate on both genes at once. The approach was to create large “double-reporter” constmcts, which consisted of a wildtype stretch of DNA containing two Hox genes, each with a different reporter gene to monitor their expression. This approach was to be used for two gene pairs: Hoxb-4/b-5, and Hoxb-l/b-2. The second reporter gene used in each case (in conjunction with LacZ) was the human placental alkaline phosphatase gene (PLAP). After initial tests of the PLAP reporter in Hoxb-1 and Hoxb-4, the latter pair (Hoxb-5/b-4) was chosen to

Fig. 4.1 Previous analysis on the regulation of Hoxb-4 and Hoxb-5.

The top panel shows the organisation of the Hoxb complex for the Hoxb-6,5 and 4 genes, and below them the stretches of DNA which have been tested for their regulatory effects. The attempt to find the important regulatory elements for Hoxb-5 is shown by the yellow bars (performed by Stefan Nonchev). Extensions were first made in a 5’ direction, and then in a 3’ direction. Only when regions E and D were included was the correct regulation found, and the two last constructs tested them independantly on a minimal Hoxb-5 region. The orange bars show the equivalent constmcts used to define the region A, B and C for Hoxb-4 (performed by J. Whiting).

Representative examples of the activity found for the regions A, C, D and E are shown below. In the central strip of images, the right-most embryo shows the pattem for activity of the Hoxb-4

promoter alone. The expression seen in the midbrain, is a well-characterised misregulation which occurs from this promoter. The panels labelled A and C show independantly the effects of region A and C (notice the midbrain expression again occuring in the second of these two constmcts). The A+B+C constmct (second down of the orange bars) recreates almost the entire Hob-4 pattem.

The bottom two photos show the neural activity of region E, and the mesodermal (plus weak neural) activity of region D, both on the Hob-5 promoter (the bottom two yellow bars).

Hoxb-6

Hoxb-5

Hoxb-4

A +B +C

r

continue the shared enhancer analysis, while the Hoxb-1 line was used for a different study described in chapter 5.

The basic experiment was as follows: First analyse the complete construct with both regulatory regions intact (regions D and E between Hoxb-5 and Hoxb-4). Then compare it with deleted constmcts in which either element has been removed. If expression in a particular tissue is lost from the patterns of both reporter genes, then the deleted region must be responsible for this aspect of regulation for both promoters. All images of Hox protein expression patterns (revealed by antibody staining) were provided by Alex Gould.