8.1 Summary of findings
I would like to introduce this Chapter with a brief resume of my findings.
Chapter 3 ~ A role fo r midline closure in the re-establishment o f dorso-ventral pattern following dorsal hindbrain ablation
From my study of neural crest regneration, it is likely that midline closure is an event with signalling properties distinct from that of simple surface ectoderm/neuroectoderm apposition. Certainly the non-head crest marker, Pax-3, was down-regulated at midline closure whilst slug was up-regulated, indicating the re-formation of the crest. The gene expression patterns were thus re-established into the normal pattern for the hindbrain; this was also true of Pax-6 expression and the branchiomotor exit points. However, re- patteming did not include all genes, Bmp-4, a candidate dorsalising factor (Liem et al.,
1995), was not re-expressed. This indicates that B m p -4 is not responsible for re- patteming and also that its expression is not regulated by the same factors that determine
slug and Pax-3 (a point not evident from the Liem et al., 1995, study).
Chapter 4 - The cloning and expression o f a twist-related gene in the chick
I demonstrated here the cloning of a rwwf-related sequence which shares many domains of expression with slug, as well as its homologues, xtwi, and mtwi (Hopwood et al.,
1989; Smith et al., 1992).
Chapter 5 - A further comparison and investigation o f the expression patterns o f slug and Pax-3 during chick einbryogenesis
The Pax-3 and slug mutually exclusive expression domains noted in the hindbrain held throughout the embryo, at least until the sixth day of chick development, and were particularly evident in the somites and their descendant lineages. In the somites Pax-3
differentiation proceeded and Pax-3 was not. This indicated that the exclusion of slug
and Pax-3 expression from the same cells was obligate, occurring at either the molecular or cellular level (direct interactions or lineage dependence).
Chapter 7 - Antagonistic ejfects o f fgf-4 and retinoic acid on slug expression in the chick limb
Slug expression marks the progress zone of the limb bud and may allow the first tentative molecular (cellular) definition of these cells. Slug was down-regulated by ridge removals or addition of medial/posterior retinoic acid bead implants; slug expression was maintained by fgf-4 protein coated beads. Hence, slug expression negatively correlated with differentiation and absence of growth but positively correlated with growth and the undifferentiated state.
8.2 Do slu g and P a x-3 define distinct lineages?
The question of lineage has been touched upon a number of times during my thesis, and overall the message was one very familiar to embryologists: pre-muscle and pre- skeletogenic cells do not transdifferentiate to each other’s fates. The investigation of this phenomenon was certianly not my intention, and it was particularly veiled at the outset during my study of the basis of neural crest cell regeneration. In fact, whilst this study constitutes the greatest part of my thesis, it was an area of the embryo in which slug and
Pax-3 expressing cells had the same origin. I was unable to definitively show that the lineage descendants of these cells were entirely non-overlapping, but the normal expression of slug and Pax-3 and the transcriptional response of the individual genes to midline closure was consistent with the cell fate changes which occurred: 5/wg-positive cells migrated and Pax-3-positive cells remained in the neural tube.
A second thread connecting the four results Chapters was a manifestation of the above statement regarding muscle and skeletal precursors, Pax-3 expressing cells differentiated into muscle or neurons and slug expressing cells into bone (eventually). A significant difference was that Pax-3 expressing cells continued to express the gene following differentiation whereas slug expressing cells down-regulated the gene prior to overt (and sometimes subtle) differentiation. This also created a potential for contradiction as whilst 5/Mg-positive cells were pre-skeletogenic, slug expression was a good marker of the undifferentiated state (e.g. Chapter 7).
What implications do these core findings have for the discussion of putative slug
function? My data regarding slug re-expression during crest regneration supports the view that slug is a candidate for the key upstream molecule that determines the epithelial to mesenchymal transition (or a number of facets of this event, see Hay, 1991). Additionally, cells expressing slug never become contractile tissue, this is a common feature of several of the j/wg-expressing populations that I have described here e.g. crest cells, sclerotome, progress zone. If this is this the result of slug expression by these cells it would indicate that the slug transcription factor functions additionally to preclude muscle differentiation pathways (directly or otherwise). There is no obvious link between these observations but one can conclude that slug expressing cells are generally uncommited but unable to give rise to muscle.
This prediction holds for the populations of cells mentioned above but is clearly not the case for all cells, e.g. at gastrulation. The expression of slug that I documented in the gastrulating mesoderm shows that most, if not all, cells express this gene, however, whilst slug is rapidly down-regulated, skeletogenesis does not occur (although some form of morphological differentiation does, e.g. epithelialisation of the notochord). Indeed as the progenitors of the myogenic blocks of the trunk (expressing Pax-3)
Nicolas et al., 1996) it appears that the adage of exclusivity has been broken. However, it is significant that these cells spend a period of time expressing neither Pax-3 nor slug,
during which time “re-programming” may occur. This suffices as an explanation for the fact that cells once expressing slug may later become muscle but highlights a potential anomaly. In Chapter 7 I have described a scenario in which slug down-regulation correlates very closely with differentiation. This is true both in the normal development of the limb (at the onset of condensation) and following experimental manipulation (retinoic acid implants and ridge removal). Thus slug is an unusual lineage marker in that it marks cells undergoing epithelial-mesenchymal transition and pre-skeletogenic cells but its down-regulation is necessary although not sufficient for further differentiation.
8.3 Reciprocity of m ig rato ry cell types in the b ran ch ial arches and lim b One feature evident from this thesis will be the reciprocity evident between the skeletogenic/musculogenic lineages in the limb and branchial arches, and the migratory partner in each structure. Much debate has centred upon the assumption of skeletogenic properties by the migratory crest, a property later regionalised to the pre-cervical crest, and the possible role of this development in vertebrate evolution (Cans and Northcutt, 1983). This is indeed a striking broadening of cell fate, but it is mirrored in the limb by the absence of myogenic potency of the cells exiting the Progress Zone, which has received considerably less attention by the evolutionists.
However, I feel in many ways it parallels the absence of the skeletogenic lineage from the head mesoderm (rostral of the notochord; Couly et al., 1993), a deficiency rectified by the abundance of ^/wg-positive crest cells. This is a population which cannot become myogenic in normal development, a feature essential for patterning (see below). Does this then reveal the causative agent of the myogenic migration to the limb? The Progress Zone comprises and maintains the proliferation of 5/wg-positive cells, which are capable
of self-renewal (Summerbell et al., 1973) but are not totipotent (cannot transdifferentiate to muscle; Duprez et al., 1996).
8.4 The role of slug and Pax-3 in segregation
Another possible role for slug can be proposed on the basis of the complementarity observed between its expression and that of P ax-3. That is that the zinc finger transcription factor encoded by slug can directly down-regulate Pax-3. It is not possible to demonstrate direct interactions between factors in vivo, although mis-expression of
slug would provide a fairly robust test - if the expansion of the slug domain led to a reduction in Pax-3 positive tissues, the slug-positive phenotype could be said to be dominant to Pax-3 (see Appendix). In Drosophila a requirement for neurogenic genes has been determined (Bate et al., 1993), which indicates that the process by which neurogenic cells are separated from the epidermis may share common features with the separation and expansion of founder myocytes from the mesoderm (and also later events - syncitial muscle differentiation). The pattern that arises is hence dependent upon this segregation, breakdown of this system, leading to transdifferentiation, causes dysmorphogenesis (Nusslein-Volhard et al., 1984).
Whilst the level of homology between Drosophila and vertebrate muscle formation is not clear, there is conservation at the gene sequence level, e.g. nautilus of drosophila (Michelson et al., 1990) and MyoD of vertebrates (Weintraub et al., 1991). Thus to draw comparisons between the strategies apparent in Drosophila and those which may occur in vertebrates has some validity. Pax-3 and slug expressing cells are often neighbours in mesodermal and neuroepithelial tissues, an environment in which segregation is essential in order to maintain distinct lineages despite exposure to many of the same signalling cues.