Mice expressing RHAG and RHD human blood group genes.

Anti-RhD prophylaxis of haemolytic disease of the fetus and newborn (HDFN) is highly effective, but as the suppressive mechanism remains uncertain, a mouse model would be of interest. Here we have generated transgenic mice expressing human RhAG and RhD erythrocyte membrane proteins in the presence and, for human RhAG, in the absence, of mouse Rhag. Human RhAG associates with mouse Rh but not mouse Rhag on red blood cells. In Rhag knockout mice transgenic for human RHAG, the mouse Rh protein is "rescued" (re-expressed), and co-immunoprecipitates with human RhAG, indicating the presence of hetero-complexes which associate mouse and human proteins. RhD antigen was expressed from a human RHD gene on a BAC or from RHD cDNA under control of ß-globin regulatory elements. RhD was never observed alone, strongly indicative that its expression absolutely depends on the presence of transgenic human RhAG. This first expression of RhD in mice is an important step in the creation of a mouse model of RhD allo-immunisation and HDFN, in conjunction with the Rh-Rhag knockout mice we have developed previously.

Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd.

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 utilises the murine Hox gene clusters encoding homeobox-containing proteins. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and might 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 reorganisation of chromosome territories and chromatin decondensation can be uncoupled from transcription itself and suggests that they can therefore operate upstream of gene expression.

Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation.

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.

Nuclear re-organisation of the Hoxb complex during mouse embryonic development.

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 Hoxb chromatin 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: Hoxb chromatin 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.

Accelerated intestinal transit in inbred mice with an increased number of interstitial cells of Cajal.

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.

Genetic interaction between a maternal factor and the zygotic genome controls the intestine length in PRM/Alf mice.

Postoperative management of small and large bowel resections would be helped by use of intestinotrophic molecules. Here, we present a mouse inbred strain called PRM/Alf that is characterized by a selective intestinal lengthening. We show that PRM/Alf intestine is one-third longer compared with other inbred strains. The phenotype is acquired mostly during the postnatal period, before weaning. Its genetic determinism is polygenic, and involves a strong maternal effect. Cross-fostering experiments revealed that the dam's genotype acts synergistically with the offspring's genotype to confer the longest intestine. Moreover, genes in the offspring have a direct effect on intestine length. Possible involvement of milk growth factors and identification of candidate genes are discussed.