The HoxTF/YYl binding-site is required for efficient transgene expression

The previously described deletion construct a5 14-606 (Figure 4.2) eliminated the C B l sequences from region C. This deletion had a profound effect on the expression pattern, compared to that derived from the wild-type region C transgene. I have shown by in vitro

analyses that the only detectable binding-site within this area is for HoxTF/YY 1. Therefore, I wished to test whether the HoxTF/YY 1 binding-site had a cw-regulatory role in transgenic mice and if its specific removal could reproduce the deletion phenotype of the a5 14-606 construct.

binding-site of the basic WT reporter construct. The cloning steps used to make this construct (MH/Y) are described in the Materials and Methods Chapter (2.3.1). The 4bp mutation introduced into the HoxTF/YYl site (GCCATTGGC to GCACGGGCC) was identical to that seen in Figure 4.5 (HoxC-M2, lane 7). When the wild-type probe (HoxC- WT) was incubated with an excess of cold mutant oligonucleotide (HoxC-MH/Y) in EMSA experiments, no competition of the HoxTF or Y Y l binding activities was observed (compare lane 5 with lanes 1 and 2 in Figure 4.6A). This confirms that at the level of sensitivity of the

in vitro assay, MH/Y represents a null mutation for HoxTF and Y Y l binding.

The MH/Y reporter construct was used to produce transgenic mice which were analysed as transient Fo embryos between 10.25 and 12.5 dpc. O f the sixteen transgenic embryos generated with this construct, ten expressed the reporter in a consistent manner and the results of this analysis are presented in Figure 4.7. The observed expression pattern was similar to that obtained with the deletion mutant a5 14-606 (Figure 4.2) with no staining present in the mesoderm. Expression was restricted to the nervous system but with a more extensive distribution than that of the a5 14-606 construct. These results show that the HoxTF/YYl binding-site within CB 1 is absolutely required for region C-directed mesodermal expression and for efficient expression of the transgene in the nervous system. Assuming that the MH/Y mutation completely abolishes the binding of HoxTF and Y Y l, as is suggested by the in vitro

studies (Figures 4.5 and 4.6), these data also indicate the presence of a further regulatory element(s), in addition to the HoxTF/YYl binding-site, that was deleted or perturbed in the a5 14-606 construct. Another possibility is that the MH/Y mutation creates a cryptic binding- site for another regulatory factor, the activity of which leads to additional expression within the nervous system in comparison to a5 14-606. These latter two points are discussed at greater length in the Summary and Discussion section (4.9) at the end of this Chapter.

At 10.25 dpc activity of the MH/Y construct was initially observed in the neural tube, running from the posterior tip to an anterior limit slightly rostral to the forelimb bud (Figure 4.7b). This boundary of neural tube expression is more posterior than that seen with the WT region C construct and the levels are much weaker. Therefore, not only is the HoxTF/YYl binding-site required for appropriate levels of expression in the nervous system, but also for attaining the proper anterior boundary of region C-directed expression at the level of the spinal cord/hindbrain boundary. By 11.5 dpc an underlying pattern of ventral staining was apparent within the neural tube with a more widespread pattern at axial levels between the limb buds (Figure 4.7c-d). Staining was particularly evident at the level of the hindlimb and was visible within the neural tube, dorsal-root ganglia and ventral motor nerves innervating the developing limb buds. However, the pattern of neural staining is patchy and represents only a subset of that specified by region C, as described in Chapter 3. At 12.5 dpc the pattern observed was similar to that seen at earlier stages but more diffuse (Figure 4.7e-f). At this

Figure 4.7: In vivo requirement for the HoxTF/YYl binding-site in C B l

The HoxTF/YY 1 site in CB 1 of region C is necessary for establishment of mesodermal and correct neural expression. The 6-galactosidase expression pattern observed in transient transgenic embryos carrying the construct MH/Y is illustrated. A schematic of the MH/Y construct and of the parental region C construct (WT) is shown below. The legend to the reporter is as described in Figure 3.3. MH/Y carries a 4bp mutation in the HoxTF/YYl site located within CB l of region C and does not bind HoxTF or Y Y l (Figure 4.6). Exp. denotes the total number of positively stained embryos obtained that showed a consistent pattern of expression. Tg. denotes the total number of transgenic embryos.

(a) Shows a lateral view of a 11.0 dpc transgenic embryo carrying the WT region C construct for comparison.

(b) Lateral view of a 10.25 dpc transgenic embryo carrying construct MH/Y. Staining can be seen in the neural tube up to a level slightly anterior to the forelimb bud. This is more posterior than that of the WT region C construct.

(c) Lateral view of an 11.5 dpc transgenic embryo carrying construct MH/Y. An underlying pattern of ventral staining can be seen within the CNS (v). A more widespread pattern of neural tube staining can be seen at levels between the limb buds, (d) Shows a transverse section through the neural tube of the same embryo, taken at the level of the hindlimb bud (lb). Patchy expression can be seen throughout the neural tube, including the floor plate (fp). Staining is also evident within the dorsal root ganglia (d) and ventral motor neuron tracts (n) of the peripheral nervous system.

(e) and (f) Show lateral and dorsal views of a 12.5 dpc transgenic embryo carrying construct MH/Y. Expression is predominantly restricted to the central nervous system with an anterior limit slightly rostral to the forelimb bud. Note the biphasic distribution of 6-galactosidase staining in the neural tube, indicated by the hollow arrows.

L

A

stage, CNS staining adopted a biphasic pattern with strong patches in the spinal cord at axial levels adjacent to the fore- and hind-limb buds. These data illustrate that the HoxTF/YYl binding-site is largely required for the activity of region C throughout its neural expression domain.

The transgenic results from the MH/Y construct clearly demonstrate that the HoxTF/YYl binding-site is a critical in vivo cw-regulatory component of region C. Although the patterns of expression observed with the MH/Y construct are not identical to those seen with the a5 14-606 deletion mutant, the effects of mutating this element in the context of the intact enhancer are widespread and indicate that it is essential for many aspects of region C-directed expression. These include activity within the mesoderm, and the levels and tissue distribution of expression within the central and peripheral nervous systems. In the context of the transcriptional regulation of the Hoxb-4 gene the role of region C activity within the nervous system is unclear, since in this respect it seems to functionally overlap with the 3'

Hoxb-4 neural enhancer region A (Whiting et a l, 1991). However, as all aspects of Hoxb-4

mesodermal expression require the activity of region C it may be logically concluded that the HoxTF/YYl binding-site is an essential component of this pattern. The results of my studies thus far have demonstrated that evolutionary sequence comparison, in conjunction with transgenic reporter gene analysis, is an useful approach for the identification of noncoding regions that function in cw-regulation. Once such regions have been identified, the use of in vitro EMSA analyses have proved valuable for the rapid identification of functional binding- sites within them.