Onset of Sek-1 expression domains

In the mouse, Sek-1 expression is first detected in a broad domain upon which the later segmental expression pattern is imposed through the up-regulation of high level expression in pre-r3 and r5, with down-regulation in pre-r4. This initial expression appears to reflect a pre-segmental subdivision within the neuroepithelium, flanked by the pre and post-otic sulci, prior to the more overt segmentation into the rhombomeres. The significance of this is unclear, in particular since it is not conserved in other species. In the avian embryo Sek-1 appears to be already expressed at high levels in presumptive r3 at the same time as the lower expression in the broad domain is up-regulated. Krox20 is first expressed in narrow stripes in pre-r3 and pre-5 in the mouse at the same time that Sek-1 is expressed in a broad domain. In Xenopus and zebrafish embryos the expression of Sek-1 reflects that of Krox20 more closely as the RNA (Xu et al., 1995) and protein are not detected in a broad domain, only in pre-r3 then pre-r5. Instead expression is detected in narrow stripes that subsequently broaden like Krox20 (Bradley et al., 1992; Oxtoby and Jowett, 1993).

This difference in expression may be due to a difference in timing in the formation of rhombomeres between these species, with specification of cells to a r3 identity occuring earlier in the chick, Xenopus and zebrafish relative to the onset of Sek-1 expression. Alternatively, there could be differences in segmental mechanisms

and/or Sek-1 function that underlie the differences in early expression. For example, Sek-1 may be part of a signal transduction cascade to demarcate early presegmental divisions in the mouse hindbrain. It will be of interest to identify the regulatory elements that direct expression to this early broad domain, and the genes responsible for Sek-1 activation here.

4.3.4 2 Broadening of r3/5 expression domains

Morphological segmentation subdivides the hindbrain into five rhombomeres of roughly equal size (r2-r6), with the segmental identity of rhombomeric cells being specified prior to segmentation (Guthrie et al., 1992). However, I find that the pre- r3/r5 domains of Sek-1 arise as narrow domains that then broaden. Sek-1 is up- regulated in pre-r3 initially in a 4-cell length domain. When pre-r3 has lengthened to 12 cells, pre-r5 is just 4 cells long, yet the definitive rhombomeres attain a similar size. Analysis of the up-regulation of Krox20 in r3 and r5 also reveals that expression is first detected in a narrow stripe that broadens, and that this broadening occurs in pre-r3 before pre-r5 while the area between the two stripes assumed to be pre-r4 changes little (Das Gupta, 1992; Irving et al., 1996). This observation extends to rhombomere formation in Xenopus and zebrafish also, as both Sek-1 and Krox20 homologues in these species are detected first in 2 narrow bands (see figure 4.c in Bradley et al., 1992; Oxtoby and Jowett, 1993). Therefore contrary to expectations that the rhombomeres might form by the simple uniform subdivision of the neural epithelium into segmental compartments of the same size, it appears that r3 and r5 arise in narrow domains that expand.

The broadening of presumptive r3 and r5 expression domains could occur by one or both of the following mechanisms. First, initial expression might occur in founder populations of pre-r3 and r5 that subsequently undergo differential cell proliferation or cell death to expand relative to even-numbered territories. Second, there may be a mechanism of progressive recruitment of cells to acquire a pre-r3 or r5 identity, that spreads rostrally and/or caudally from the initial narrow domain of

expression. Sek-1 expression does appear to spread from the lateral neural plate medially within the pre-rhombomeric domains, suggestive of a wave of up-regulation along this axis. The observation of progressive growth of each rhombomere from an initially small group of cells raises the possibility that the pre-r3/r5 gene expression in narrow domains reflects a specification through short range interactions.

Unlike the mouse, in the chick Cek8 and Krox20 (Nieto, A. et al., 1995) expression in presumptive r3/5 arise in broad domains, rather than narrow domains that then broaden. It is possible that the initial specification of presumptive r3 and r5 in narrow domains is a feature of mouse development that does not occur in the chick, Xenopus and zebrafish. Alternatively, this difference may be accounted for by timing differences between the formation of the rhombomeres and the onset of expression in these species. In the chick the presumptive rhombomere domains may have already broadened before Cek8?mù Xro%20are expressed. The identification of earlier markers of rhombomere specification may enable these possibilities to be distinguished.

Sek-1 becomes up-regulated in presumptive r3 and pre-r5 at the same time that Krox20 transcripts are seen in the same regions. Thus these genes may be part of a regulatory cascade for up-regulation of expression in r3 and r5. Krox20 has already been identified as the gene responsible for up-regulation of Hoxb-2 in r3 and r5 (Sham et al., 1993). Alternatively, Sek-1 may be involved in a signal transduction pathway to up-regulate Krox20. Future experiments looking for Krox20 binding sites in the 5’ regulatory region of Sek-1, and analysis of the expression of Krox20 in response to activation of the Sek-1 receptor will provide further insights into a possible regulatory relationship. Recent data from Dr. T. Theil in our lab indicates that Krox20 does indeed regulate the r3/r5 expression of Sek-1.

It is interesting to note that the expression pattern of XSek-1 and rtkl so closely mimics that of XKrox20 and zebrafish Krox20. In these species it is possible that Krox20 is directly responsible for the activation of these genes in the hindbrain, whereas in mouse it is likely that another gene is responsible for the initial rhombomeric expression in a broader area.

4.3.43 Progressive sharpening of expression domains

Between 8 and 12 somites in the mouse embryo the boundaries of the high level domains of expression in pre-r3 and pre-r5 are fuzzy, with groups of cells protruding from the main domains of expression, and some isolated individual cells expressing Sek-1 protein close to the main body of expression. These cells probably do not represent a particular cell type as the pattern is inconsistent between different embryos, and between the left and right halves of the rhombomere. Rather, these data support the idea of dynamic gene regulation during the establishment and maintainance of Sek-1 expression. Sharpening of the expression domains of Sek-1 as the rhombomere boundaries form may occur by one or both of two mechanisms: lineage restriction and cell fate switching, which I will discuss below.

As the rhombomere boundaries form and cellular differences become apparent between cells of neighbouring rhombomeres, sharpening of the expression domains may be as a result of these alternating properties of rhombomeric cells. Cellular differences between adjacent rhombomeres have only been observed after segmentation, but it is likely that they are progressively established and lead to a segregation of cells with alternating odd and even properties that subsequently lead to the sharpening of gene expression domains. The observed expression patterns of both Sek-1 and Krox20 correlate with such a mechanism as their domains of expression become increasingly sharp during the period that the rhombomeres differentiate and display alternating properties.

Analyses of clonal restriction in the hindbrain during segmentation of the neural epithelium reveals that the progeny of cells marked before rhombomere formation disperse considerably and can contribute to adjacent rhombomeres as discussed in chapter one (Fraser et al., 1990). This dispersal may be due to the intercalation of other cells during cell division leading to the separation of clonally related progeny (Kimmel et al., 1994). The initial diffuse domains of Sek-1 expression in pre-r3/r5 are consistent with the mixing of cells between presumptive rhombomeres. However, the expression domains are remarkably precise considering that clones may disperse over a rhombomere length in the chick (Fraser et al., 1990) and that there is still movement of cells between rhombomeres after boundary

formation (Birgbauer and Fraser, 1994) when high level Sek-1 expressing cells are tightly restricted within r3 and r5. The stripes of expression of KroxIO sharpen into discrete domains during rhombomere boundary formation with occasional ‘violating’ cells expressing Krox20 in an even-numbered rhombomere (Das Gupta, 1992; Irving et al., 1996). The progressive sharpening of expression is very similar to that of Sek-1 in the same rhombomeres.

The spatial restriction of expression in this environment can be explained by a community effect model where cell identity is regulated to be the same as neighbouring cells by local interactions within a presumptive rhombomere. The community effect model predicts that any cells moving between presumptive rhombomeres encounter short range signals that cause them to switch identity to that of the cells now surrounding them. An alternative possibility is that all cells crossing between presumptive rhombomeres die rather than switch fate. Clonal analysis however, does not support this hypothesis as clonal progeny can make a major contribution to adjacent rhombomeres (Fraser et al., 1990). Most clonal progeny are restricted to a single rhombomere after segmentation but even at this stage some cells are still detected crossing rhombomere boundaries (Birgbauer and Fraser, 1994). These cells may correspond to those occasional cells detected expressing Sek-1 at a high level in an even-numbered territory. At present it is not possible to tell if these cells are in the process of changing fate to that of their new environment. In the future cell marking experiments coupled to immunodetection may clarify this.

Indirect support for this model of dynamic regulation of cell fate comes from experiments in the mouse that identified an r4 enhancer and r3/r5 repressor element that restricts Hoxb-1 expression to r4 (Studer et al., 1994). In constructs containing Hoxb-1 regulatory sequences that included the repressor, expression of a reporter gene is tightly restricted to r4. However, in the absence of this repressor, gene expression spreads from r4 into the adjacent rhombomeres: r3 and r5. The reporter gene expression in r3 and r5 may correspond to cells that were transiently specified as pre-r4, but failed to repress expression as they moved into the adjacent compartment. These experiments also provide further support that cell death is not important for restricting gene expression, at least at these early stages examined. Conclusive evidence for this proposed model might be obtained by combining lineage

tracing with gene expression analysis and by transplantation of individual cells between rhombomeres, to determine whether cells are ever irrevocably committed or if their segmental identity is fluid and regulated by local interactions.

The relative contributions of a dynamic regulation of cell identity and the restriction of cell movement between odd and even presumptive segments is unknown, and may change as the rhombomeres are established. It is likely that both mechanisms are important in segmentation of the rhombomeres, and also in the establishment and maintainance of the precise expression domains of Sek-1.

43.4.4 The role of Sek-1 in hindbrain segmentation

Several studies have indicated the roles of Sek-1 and Krox20 in patterning rhombomeres 3 and 5. The Krox20 gene is required for the formation of r3 and r5 (Schneider-Maunoury et al., 1993; Swiatek and Gridley, 1993) and for the expression of Hoxb-2 in these rhombomeres (Sham et al., 1993), and therefore its segment- restricted expression may be critical for the specification of r3 and r5. Disruption of Sek-1 function in Xenopus and zebrafish using a dominant negative approach results in changes in the domains of expression of Sek-1 in the hindbrain; Sek-1 expression is not restricted to r3 and r5 but spreads into the adjacent r2, r4 or r6 (Xu et al.,

1995). This expression may reflect cells that have crossed the rhombomere boundary but are unable to change fate to that of an even-numbered rhombomere because of a block in cell signalling caused by the Sek-1 mutant, resulting in the maintainance of expression of the odd-numbered rhombomere gene marker. Alternatively, Sek-1 may have a role in the restriction of cell movement between rhombomeres, such as mediating contact-dependent repulsion of odd-numbered rhombomere cells by even- numbered ones. Taken together, these data indicate that Krox20 and Sek-1 have key roles in the processes that I have proposed regulate the expression domains. Krox20 up-regulates expression of Sek-1 in pre-r3 and pre-r5, and this receptor mediates either the switching of cell identity or the restriction of cell movement. As a consequence, expression of both genes is either maintained or sharpened during

segmentation.

4.3.5 Lineage restriction and cell identity switching in Drosophila

The models that I am proposing for segmentation in the hindbrain have interesting similarities to events during parasegment formation in Drosophila. A photoactivated lineage tracer was used to follow the progeny of single cells from the blastoderm stage, whilst monitoring their expression of engrailed (Vincent and O ’Farrell, 1992). en is expressed in one row of cells in the posterior part of each parasegment in the Drosophila embryo, and is required to maintain cell fate. Clonal analysis revealed that no clones straddled the anterior boundary of the en stripe (and therefore the parasegment boundary) revealing that clonal restriction is established here before germband extension when the embryos were examined. Posterior to the boundary however, clones were found to be a mixture of en expressing cells adjacent to the boundary and non-expressing cells that spread posteriorly within the parasegment. The expression of en is maintained by short-range signalling of wingless (wg), which is expressed in the anterior most row of cells in each parasegment and therefore next to the en expressing cells in the adjacent parasegment. As the progeny of en- expressing cells move away from the parasegmental boundary they no longer receive the wg signal and therefore down-regulate en expression. Thus these cells are not permanently committed to express en but are subject to a plasticity of cell fate, dependent on the local cellular environment at this stage (Vincent and O ’Farrell, 1992 and reviewed in DiNardo et al., 1994).

CHAPTER FIVE