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The restriction of cells to individual compartments allows each compartment to maintain a unique identity. This can be observed in the hindbrain during the formation of the rhombomeres and an important question is how this is established.

1.5.1 Compartments and boundaries.

The restriction of cell movement between rhombomeres could be due to intrinsic differences between adjacent segments, or the formation of a barrier at rhombomere boundaries. In support of the latter possibility, rhombomere boundaries contain cells which have distinct characteristics to cells in the centre of rhombomeres (Heyman et al., 1995). These boundary cells have different rates of cell division and unlike most cells of the neural epithelium and do not undergo interkinetic nuclear migration during cell division (Guthrie et al., 1991), in which the nuclei of dividing cells migrate basally as the cells go through S- phase in the cell cycle and subsequently return to the apical surface of the cell. They are therefore a relatively static population of cells. The extracellular space is increased between boundary cells with higher levels of chondroitin sulphate proteoglycan in the extracellular spaces (Heyman et a i, 1995). The rhombomere boundary cells also show differences from the rhombomere cells themselves in patterns of gene expression. There is an increase in levels of the intermediate filament protein, vimentin, at the rhombomere boundaries (Heyman et al., 1995) along with increased levels of NgCAM (Lumsden and Keynes, 1989) and peanut lectin (Layer and Alber, 1990). This pattern of gene expression may indicate a radial glial phenotype for these cells (Guthrie, 1996). In mature rhombomere boundaries there is a decrease in expression of Krox20 and Hox-bl. Correspondingly there is an increase in expression of PLZF, Fgf-3, and Pax-6 only in the boundaries (Heyman et al., 1995). The existence of these distinctive boundary cells raises the possibility that they act as a barrier to prevent intermingling between rhombomeres.

Another possibility is that differences in the cell surface properties of cells from different rhombomeres prevent cell mixing. To test this hypothesis donor rhombomeres lacking their boundaries have been transplanted so that rhombomeres from different anteroposterior levels are juxtaposed (Guthrie and Lumsden, 1991). In this way it was demonstrated that when even numbered rhombomeres are placed next to odd numbered rhombomeres the boundaries reform, but when two even or odd numbered rhombomeres are juxtaposed boundaries do not re-establish themselves. In other experiments, rhombomere fragments grafted into various positions along the anteroposterior axis in the chick have been used to

show that when even numbered rhombomeres are combined with cells from other even numbered rhombomeres, extensive cell mixing occurs (Guthrie et a l, 1993). Similarly, cell mixing also occurs when odd numbered cells are placed in odd numbered rhombomeres, but to a lesser degree. When even numbered tissue is placed in an odd numbered rhombomere, however, the transplanted cells do not intermingle with the cells of the recipient rhombomere. These phenomena suggest that there are differences in cell surface and/or adhesion properties in neighbouring rhombomeres that serves to restrict cell movement and points to a two segment periodicity model in hindbrain segmentation.

1.5.2 Segmentation of neural crest.

In the chick and the mouse, streams of cranial neural crest migrating to the first, second, and third branchial arches are each separated by neural crest free zones that lie opposite to rhombomeres 3 and 5. Experiments in the chick have demonstrated that there are increased levels of cell death in rhombomeres 3 and 5 and it has been suggested that this may account for the crest free zones (Lumsden et al., 1991) (Jeffs et al., 1992). Rhombomere explant experiments have shown that the increased levels of cell death in rhombomeres 3 and 5 are caused by the presence of the adjacent even numbered rhombomeres since rhombomeres 3 and 5 produce migrating neural crest cells when explanted alone (Graham et al., 1993). It has also been demonstrated that even numbered rhombomeres are responsible for the maintenance of expression of msx2 in rhombomeres 3 and 5. msx2 expression has been shown to correlate with increased levels of apotosis (Graham et al., 1993). Moreover, it has been shown that BMP4 expression in rhombomeres 3 and 5 is also dependent on the presence of even numbered rhombomeres. Addition of recombinant BMP4 protein to isolated explant cultures of either rhombomere 3 or 5 upregulate the expression of msx2

and suggest that the levels of apoptosis are also increased (Graham et al., 1994). These experiments suggest that rhombomeres 3 and 5 do not produce neural crest cells because they are eliminated by apoptosis through the action of msx2 through BMP4 (Lumsden et a l, 1991).

Lineage tracer injections into individual rhombomeres, however, have revealed that rhombomeres 3 and 5 do produce a small number of neural crest cells (Sechrist et a l,

1993) which deviate either rostrally or caudally to join the streams of migrating crest emanating from the adjacent even numbered rhombomeres (Birgbauer et al., 1995). Analysis of the timing of cell death in rhombomeres 3 and 5 have shown that it occurs after the neural crest cells have migrated and is thus unlikely to account wholly for the neural

crest free zones opposite rhombomeres 3 and 5 (Sechrist et al., 1993). It has been shown that rhombomere 3 ceases to produce neural crest cells earlier than other rhombomeres (Sechrist et al., 1993) and that the neural crest cells also preferentially exit near the rhombomere 3/4 border when rhombomere 3 is transplanted to the position of rhombomere 4 indicating either an attractive exit point or inhibitory signals in rhombomere 3 (Sechrist et al., 1994). This may account for the crest free zones opposite rhombomere 3. When rhombomere 4 is transplanted to the position of rhombomere 3 it has been shown that neural crest cells enter the mesenchyme adjacent to rhombomere 3 (Sechrist et al., 1994) which is normally devoid of neural crest cells. This indicates that although the mesenchyme adjacent to rhombomere 3 is normally inhibitory to neural crest cells from rhombomere 3 it is not inhibitory to neural crest cells emerging from rhombomere 4. In experiments where otic placodes are transplanted neural crest cells are observed migrating towards the ectopic otic vesicle, suggesting the presence of a chemoattractant (Sechrist et al., 1994). Ablation or partial ablation of the otic vesicle alters the pattern of neural crest cell migration such that labelled rhombomere 4 neural crest cells contribute to the third branchial arch as well as the second branchial arch (Sechrist etal., 1994). Together, these suggest that in addition to the presence of the otic vesicle being a physical barrier that blocks neural crest cell migration opposite rhombomere 5 and rhombomere 6 , it may also have an active role in guidance

(Sechrist et al., 1994).