3.3.4) Therefore Msx-1 and Msx-2 are differentially expressed between

atrio-ventricular and outflow tract cushions as they are expressed throughout the atrio­ ventricular cushion from the earliest stage o f cushion formation. By E l 1.5, however, expression within the outflow tract cushions is up-regulated and equivalent to that detected within the atrio-ventricular cushions (see section 3.3.3; 3.3.4).

As described for the initial stages o f cushion formation, the Msx-2 expression data

in mouse presented in this thesis are contrary to that previously described in chick (Chan- Thomas et al., 1993). In the developing chick embryo, Msx-2 is not reported to be

detected within the developing cushions during expansion, but is predominantly restricted to the atrio-ventricular myocardium. There are now a number o f genes that have been

reported to have different expression patterns in chick and mouse, for example TG Fpi-3

(see Table 1.1). These differences are presumably the result o f the time elapsed since mammals and avians diverged.

3.9.3

Interaction of BM P and M sx genes during cushion formation

the forming and expanding outflow tract endocardial cushions (see section 3.3.2.1), which themselves express Msx-1 and Msx-2 (see sections 3.3.3; 3.3.4). Similarly, in the atrio­

ventricular region, BMP-2 is expressed within the myocardium underlying cushion tissue

(see section 3.3.1.1), which also expresses Msx-1 and Msx-2. This close association o f

BMP and Msx genes in adjacent tissues has been documented in many developing

systems. The interactions o f Msx and BMP genes during development has been studied in

the limb (Ferrari et al., 1998), mammary gland (Phippard et al., 1996) and tooth (Bei and Maas, 1998; Tucker et al., 1998), and during the regulation o f neural crest cell apoptosis (Graham et al., 1994; Takahashi et al., 1998). The interactions o f BMP-4 and Msx-1

within the developing tooth are, in many respects analogous to the situation seen within the developing outflow tract, with the myocardium corresponding to the dental epithelium and the cushion tissue to the branchial arch mesenchyme. During initiation o f tooth formation at E l0.5, BMP-4 expression is detected within the dental epithelium overlying

the neural crest-derived mesenchyme o f the branchial arch (Vainio et al., 1993), which expresses Msx-1 (Chen et al., 1996). At this stage in tooth development, BMP-4 has been

shown to regulate mesenchymal Msx-1 expression. Removal o f the 5MP-4-positive

dental epithelium results in down-regulation o f Msx-1 expression, and upon addition o f

BMP-4 soaked beads, Msx-1 expression is restored (Tucker et al., 1998; Bei and Maas,

___________________________________________________________________________Chapter Three

dental epithelium also acts to down-regulate the underlying mesenchymal expression o f

Msx-1.

The situation by E l 1.5 in the developing tooth is quite different, however. At this stage, BMP-4 and Msx-1 are both expressed within the underlying mesenchyme,

concurrent with a down-regulation o f BMP-4 within the dental epithelium. The up-

regulation o f BMP-4 within the mesenchyme is thought to be dependent on Msx-1

expression. This has been inferred from the Msx-1 knock-out mouse, as mesenchymal

expression o f BMP-4 is lost in El 1.5 Msx-l-mx\\ embryos (Chen et al., 1996).

This spatio-temporal pattern o f BMP-4 and Msx-1 expression directly correlates

with the expression patterns detected within the outflow tract myocardium and cushion tissue described in this Chapter. It is possible that a similar interdependent regulatory mechanism operates within the atrio-ventricular region, involving BMP-2 and Msx-1 and

or Msx-2 genes.

3.9.4

Tenascin-C is down regulated at E10.5, and subsequently up-

regulated at E l 1.5 in the atrio-ventricular cushions

Tenascin-C has the most diverse expression patterns during heart development o f the

genes investigated in this thesis (see sections 3.3.6- 3.3.6.2). Expression is detected within the atrio-ventricular and outflow tract transforming cushion cells from E9.5 to E l0.5, but expression is down-regulated in the atrio-ventricular cushions, although maintained within the outflow tract cushions.

Tenascin-C is thought to be active at sites o f epithelial to mesenchymal

transformation, promoting cell-substrate adhesion (Hurle et al., 1990; Crossin and Hoffman, 1991) (see section 1.5.6; 1.5.6.1). Therefore, the down-regulation o f tenascin-

o f epithelial to mesenchymal transformation from the endocardial cells had slowed or ceased. It is possible that mesenchymal cell proliferation, rather than the continued recruitment o f new cells from the endocardium, results in the expansion o f the atrio­ ventricular cushions. Despite the fact that the outflow tract cushions are developmentally retarded by 12 hours compared with the atrio-ventricular cushions, tenascin-C continues

to be expressed for longer in the outflow tract cushion than the atrio-ventricular cushions. This may suggests a need for continued recruitment o f endocardial cells within the outflow tract in comparison to the atrio-ventricular cushions, or a need for the population o f the outflow tract with neural crest cells.

3.9.4.1

Tenascin-C

regulated in the atrio-ventricular cushion at £11.5

The up-regulation o f tenascin-C within the superior atrio-ventricular cushion at E l 1.5 as

well as the maintenance o f tenascin-C expression within the outflow tract cushions and

myocardium (see section 3.3.6; 3.3.6.1), may be regulated by an entirely different mechanism. The spatio-temporal patterns o f tenascin-C protein within the developing chick outflow tract after cushion formation have been interpreted by Hurle and colleagues (1990) to correlate with the sites o f maximum mechanical stress, due to the myocardial contractions and haemodynamic forces acting on the outflow tract. Moreover, tenascin-C

has been shown to be up-regulated in cultured neonatal myocytes when mechanical strain is applied in the form o f a deformation o f the culture substrate (Yamamoto et al., 1999) (see section 1.5.6.1). With the dramatic growth o f the embryo by E l 1.5 and the

increasingly larger volume o f blood pumping though the heart, the haemodynamic forces may be strong enough to induce mechanical strain within the heart. This phenomenon could explain the maintained tenascin-C expression within the endocardial cushion and

Chapter Three

myocardium o f the developing mouse outflow tract, and perhaps the novel expression described in this Chapter within the superior atrio-ventricular cushion at E l 1.5 (see section 3.3.6).

3.10 A ROLE FOR BMP-2 AND BMP-4 IN THE INDUCTION OF