The relocalisation of some genes to positions outside chromosome territories, and the visible decondensation or unfolding of interphase chromatin, are two striking facets of nuclear reorganisation linked to gene activation that have been assumed to be related to each other. Here, in a study of nuclear reorganisation around the Hoxd cluster, we suggest that this may not be the case. Despite its very different genomic environment from Hoxb, Hoxd also loops out from its chromosome territory, and unfolds, upon activation in differentiating embryonic stem (ES) cells and in the tailbud of the embryo. However, looping out and decondensation are not simply two different manifestations of the same underlying change in chromatin structure. We show that, in the limb bud of the embryonic day 9.5 embryo, where Hoxd is also activated, there is visible decondensation of chromatin but no detectable movement of the region out from the chromosome territory. During ES cell differentiation, decondensed alleles can also be found inside of chromosome territories, and loci that have looped out of the territories can appear to still be condensed. We conclude that evolutionarily conserved chromosome remodelling mechanisms, predating the duplication of mammalian Hox loci, underlie Hox regulation along the rostrocaudal embryonic axis. However, we suggest that separate modes of regulation can modify Hoxd chromatin in different ways in different developmental contexts.

The spatial and temporal co-linear expression of Hox genes during development is an exquisite example of programmed gene expression. The precise mechanisms underpinning this are not known. Analysis of Hoxbchromatin structure and nuclear organisation, during the differentiation of murine ES cells, has lent support to the idea that there is a progressive`opening' of chromatin structure propagated through Hox clusters from 3′to 5′, which contributes to the sequential activation of gene expression. Here, we show that similar events occur in vivo in at least two stages of development. The first changes in chromatin structure and nuclear organisation were detected during gastrulation in the Hoxb1-expressing posterior primitive streak region: Hoxbchromatin was decondensed and the Hoxb1 locus looped out from its chromosome territory, in contrast to non-expressing Hoxb9, which remained within the chromosome territory. At E9.5, when differential Hox expression along the anteroposterior axis is being established, we found concomitant changes in the organisation of Hoxb. Hoxb organisation differed between regions of the neural tube that had never expressed Hoxb [rhombomeres (r) 1 and 2], strongly expressed Hoxb1 but not b9 (r4), had downregulated Hoxb1 (r5), expressed Hoxb9 but not Hoxb1 (spinal cord), and expressed both genes(tail bud). We conclude that Hoxb chromatin decondensation and nuclear re-organisation is regulated in different parts of the developing embryo, and at different developmental stages. The differential nuclear organisation of Hoxb along the anteroposterior axis of the developing neural tube is coherent with co-linear Hox gene expression. In early development nuclear re-organisation is coupled to Hoxb expression,but does not anticipate it.

Summary The extent to which the nuclear organisation of a gene impacts on its ability to be expressed, or whether nuclear organisation merely reflects gene expression states, remains an important but unresolved issue. A model system that has been instrumental in investigating this question is the murine Hox clusters. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and may be linked to mechanisms underlying colinearity. To investigate this, and to delineate the cis-acting elements involved, here we analyse the nuclear behaviour of a 3' Hoxb1 transgene transposed to the 5' end of the Hoxd cluster. We show that this transgene contains the cis-acting elements sufficient to initiate ectopic local nuclear reorganisation and chromatin decondensation, and to break Hoxd colinearity, in the primitive streak region of the early embryo. Significantly, in rhombomere 4 the transgene is able to induce attenuated nuclear reorganisation and decondensation of Hoxd even though there is no detectable expression of the transgene at this site. This shows that chromosome territory reorganisation and chromatin decondensation can be uncoupled from transcription itself, and suggests that they can therefore operate upstream of gene expression.

The interstitial cells of Cajal (ICC) play an important role in coordinating intestinal motility, and structural alterations in ICC are found in several human digestive diseases. Mouse models with defects in ICC allow a better understanding of their functions. We investigated the pattern of intestinal motility and the distribution of ICC in the PRM/Alf inbred mouse strain, characterized by a selective intestinal lengthening. In PRM/Alf mice, the digestive transit time, evaluated by using thermophilic Bacillus subtilis spores, was normal, indicating accelerated transit. The contractility and slow-wave frequency, recorded on isolated segments from the proximal small intestine, were significantly increased. The number of ICC was also significantly higher along the small intestine and the colon. The concomitant increase of the contractility, the slow-wave frequency, and the number of ICC is consistent with the proposal of a role of ICC number increase in the higher intestinal transit speed. The PRM/Alf model should be useful to further investigate the roles of ICC in the control of digestive motility. gut length; kit receptor; electrical slow waves; gastrointestinal motility; genetic model INTRINSIC AND EXTRINSIC NEUROHUMORAL signals control intestinal motility. An essential part of the system is the electrical pacemaker activity that originates in interstitial cells of Cajal (ICC). These cells are present in close association with smooth muscle cells and neurons of the gastrointestinal tract. In the mouse, ICC are fully differentiated at weaning, when the animals are given adult diet (13). The possible role of ICC in coordinating the contractile activity of the intestine has come to light in recent years (17,38). Alterations of ICC were reported in a variety of gastrointestinal disorders, including hypertrophic pyloric stenosis (24, 35, 41), Hirschsprung's disease (37,42,43), and intestinal pseudo-obstructions (19,21,44). Study of mouse models has proven to be a valuable strategy for studying the cause-and-effect relationship between ICC and motility problems, and Kit/W and SCF/Steel mutant mice have been instrumental in the study of the physiological roles of ICC (reviewed in Ref. 30). The Kit signaling pathway is essential for the development and maintenance of ICC (26). Both Kit and Steel mutant mice exhibit absence of electrical rhythmicity (i.e., slow waves) associated with underdevelopment of certain classes of ICC, including ICC of the small intestine that lie in the plane of the myenteric plexus (ICC-MP) between the circular and longitudinal muscle layers (17,38,39). The absence of ICC and slow waves in the small intestine of Kit mutant mice was paralleled with altered peristaltic movements of intestinal contents (10). However, it remains difficult to predict how abnormalities in ICC could influence digestive motor activity in vivo, because peristalsis is controlled by multiple, simultaneously operating mechanisms, in particular within the enteric nervous system (34). Indeed, migrating motor complexes can b...