APLICACIÓN DEL MODELO COMUNITARIO PROPUESTO EN EL

The introductory aim of this project was to assemble a region C reporter construct which could be employed in transgenic mice. This would be used to further define the developmental expression pattern dictated by region C. Having determined a baseline, smaller cw-regulatory sequences could be delimited by comparing the expression pattern derived from mutant constructs to that of the wild-type.

The cloning steps used to generate the wild-type region C construct (WT) are described in the Materials and Methods Chapter (2.3.1). WT comprises region C cloned upstream of a reporter construct consisting of the hsp68 promoter driving lacZ, with the SV40 termination and polyadenylation sequences attached to the 3' end (Whiting et a l, 1991). The hsp68

promoter was employed for several reasons. Firstly, region C is able to confer spatial- specificity on the hsp68 promoter and impose an expression pattern on it very similar to that seen with the Hoxb-4 promoter. Secondly, it provides relatively high levels of reporter gene expression compared to those seen with the Hoxb-4 promoter. It was envisaged that this would be advantageous for the analysis of mutant constructs where low levels of expression might be expected. Thirdly, the heterologous promoter provides a context in which to study region C enhancer activity in isolation from other regulatory elements that are known to be present within the Hoxb-4 promoter (Gutman et a l, 1994).

WT was microinjected into the pronuclei of one-cell mouse embryos as described in the Materials and Methods Chapter (2.3.3). Transgenic mice generated in this way were initially

examined by transient analysis of Fo embryos at 10.5 days post coitum {àpc). This was the developmental time point previously described for the expression of an equivalent construct (Whiting et a l, 1991). Of the six transgenic embryos obtained, all four that expressed the transgene did so in a manner consistent with that previously described (data not shown). These data indicated that WT was functional and capable of giving appropriate region C- directed expression. In addition to the transient analysis a transgenic line was established which was mated to wild-type animals to obtain embryos at various developmental time- points. This enabled the expression pattern derived from this region C reporter to be studied throughout embryogenesis, which has not been previously done. The results of these analyses are shown in Figure 3.3.

Transgene expression driven by region C (WT) was initially activated in the late pre-somite stage embryo at 7-7.5 dpc. Staining due to 6-galactosidase activity was detectable within the ectoderm, mesoderm and endoderm posterior to the neural groove, and also at the base of the allantois (Figure 3.3a). At 8.5 dpc (10-12 somite stage) strong 6-galactosidase staining was visible within the posterior neuroectoderm and neural tube, then more weakly up to an anterior limit at the presumptive spinal cord/hindbrain boundary (Figure 3.3b). This is more posterior than the normal boundary of endogenous H o x b -4 expression within the neuroectoderm which extends to the level of the r6/7 boundary (Graham et a l, 1988; Whiting

et a l, 1991). Mesodermal staining was widespread and located in the somitic, presomitic and lateral mesoderm. Somitic staining was visible in the most posterior, newly formed, somite up to an anterior limit at the level of s6/7. In a analogous manner to the neural expression pattern the most anterior somites were much more weakly stained.

The expression pattern observed at early stages was maintained throughout later stages of development. Between 9.5 and 12.5 dpc strong staining could be clearly seen in the neuroectoderm and neural crest derivatives, such as the spinal ganglia, and in the somitic and lateral mesoderm, posteriorly from the forelimb (Figure 3.3c-g). Anterior limits of expression in the spinal cord and somites remained weak, however, becoming stronger more posteriorly. This is somewhat different from the pattern observed when region C is used to drive expression from the Hoxb-4 promoter. In this case the levels of expression are strongest at the anterior boundaries, becoming weaker more posteriorly. By 15.5 dpc, 6- galactosidase detectability is impared due to the developing skin acting as a barrier to the fixation and staining processes. At this stage, staining could be seen in the developing mammary glands (data not shown) and in the primordia of sensory hair follicles of the face (Figure 3.3h), located above the eye (Kaufman, 1992). This particular aspect of follicular expression is different from that seen with the full length Hoxb-4-lacZ transgene previously described (Whiting et a l, 1991). In this latter case 6-galactosidase activity was noted only in the dermal placodes of the skin with an obvious axial limit at the cervical level.

Figure 3.3: Developmental time-course of region C/hsp68-lacZ reporter gene expression

A time-course of transgenic embryos carrying the mouse Hoxb-4 region C enhancer within construct WT is illustrated. A schematic of the WT reporter is shown below. The hsp68-lacZ-

SV40pA reporter gene is not to scale. Major restriction enzyme sites are marked and the legend to region C follows that described in Figure 3.1 and Figure 4.1. Exp. denotes the total number of positively stained lines (1) and transient embryos (4) obtained that showed a consistent pattern of expression. Tg. denotes the total number of transgenic founders and embryos.

(a) Lateral view of a 7.5 dpc transgenic embryo showing staining in the ectoderm, mesoderm and endoderm posterior to the caudal tip of the neural groove (arrowhead). The anterior end of the embryo is to the left (a) and the posterior end to the right (p). (b) Dorsal view of an 8.5 dpc

embryo, with the anterior end at the top of the picture. Strong staining can be seen in the posterior neuroectoderm and neural tube, extending to an anterior limit at the presumptive spinal cord/hind brain boundary. Note that the levels of neural tube staining at the anterior boundary are weak. Widespread staining is also visible within the lateral, presomitic and somitic mesoderm. Somitic staining is similarly weak towards its anterior margin at the boundary between somites 6 and 7 (s6 and s7). Only feeble staining is visible in s7, indicated by an open arrowhead. The boundaries between somites are indicated by arrows, (c) Lateral view of a 9.5 dpc transgenic embryo, by which stage the anterior somite boundary is clearly visible at the border between s6 and s7 (arrow), (d) and (e) Lateral and dorsal views of a 10.5

dpc transgenic embryo. An anterior limit of strong staining within the neural tube is visible (>), progressing much more weakly up to the spinal cord/hindbrain boundary marked by an arrow, (f) A transverse section through the forelimb bud (lb) of a similar 10.5 dpc embryo (lOX magnification), where staining can be seen in the somite (s) and in the neural tube (nt).

(g) Lateral view of a 12.5 dpc embryo, (h) Lateral view of the head of a 15.5 dpc embryo. Staining can be seen in the primordia of the follicles of vibrissae around the snout (hf) and in the prominent tactile hair follicles (>).

WT