XCI in preimplantation mouse and human embryos: first there is remodelling .

Female eutherians silence one of their X chromosomes to accomplish an equal dose of X-linked gene expression compared with males. The mouse is the most widely used animal model in XCI research and has proven to be of great significance for understanding the complex mechanism of X-linked dosage compensation. Although the basic principles of XCI are similar in mouse and humans, differences exist in the timing of XCI initiation, the genetic elements involved in XCI regulation and the form of XCI in specific tissues. Therefore, the mouse has its limitations as a model to understand early human XCI and analysis of human tissues is required. In this review, we describe these differences with respect to initiation of XCI in human and mouse preimplantation embryos, the extra-embryonic tissues and the in vitro model of the epiblast: the embryonic stem cells.

Defective deacetylation of histone 4 K12 in human oocytes is associated with advanced maternal age and chromosome misalignment.

BACKGROUND: Chromosome segregation errors during human oocyte meiosis are associated with low fertility in humans and the incidence of these errors increases with advancing maternal age. Studies of mitosis and meiosis suggest that defective remodeling of chromatin plays a causative role in aneuploidy. We analyzed the histone deacetylation pattern during the final stages of human oocyte maturation to investigate whether defective epigenetic regulation of chromatin remodeling in human oocytes is related to maternal age and leads to segregation errors. METHODS: Human surplus oocytes of different meiotic maturation stages [germinal vesicle (GV), metaphase (M)I and MII] were collected from standard IVF/ICSI treatments. Oocytes were analyzed for acetylation of different lysines of histone 4 (H4K5, H4K8, H4K12 and H4K16) and for α-tubulin. RESULTS: Human GV oocytes had an intense staining of the chromatin for all four histone 4 lysine acetylations. MI and MII stage oocytes showed either normal deacetylation or various amounts of defective histone deacetylation. Residual H4K12 acetylation was more frequently found in oocytes obtained from older women, with a significant correlation between defective deacetylation and maternal age (r = 0.185, P = 0.007). Eighty-eight percent of the oocytes with residual acetylation had misaligned chromosomes, whereas only 33% of the oocytes that showed correct deacetylated chromatin had misaligned chromosomes (P < 0.001). CONCLUSIONS: We conclude that defective deacetylation during human female meiosis increases with maternal age and is correlated with misaligned chromosomes. As chromosome misalignment predisposes to segregation errors, our data imply that defective regulation of histone deacetylation could be an important factor in age-related aneuploidy.

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.

Neurohypophysial dysmorphogenesis in mice lacking the homeobox gene Uncx4.1.

A number of transcription factors have been implicated in the development of the hypothalamo-neurohypophysial system (HNS). Null mutations for these factors caused severe defects in proliferation, migration and survival during early embryogenesis. While they have informed about early events of HNS developments no insights in mechanisms of late development and maturation of this major peptidergic system have been obtained as yet. In a screen for adult-expressed homeobox genes we identified Uncx4.1 as a gene expressed in adult and embryonic magnocellular neurons of the (HNS). Null mutation of Uncx4.1 left these neurons viable and able to express neuropeptides. However, the connectivity of magnocellular neurons with posterior pituitary elements was compromised. As a consequence neuronal fibres traversed to the adenohypophysis. The penetrance of this phenotype was about 50%. The data show a selective role of Uncx4.1 in controlling the development of connections of hypothalamic neurons to pituitary elements, allowing central neurons to reach the peripheral blood circulation and to deliver hormones for control of peripheral functions.

GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei.

The GATA-3 transcription factor shows a specific and restricted expression pattern in the developing and adult mouse brain. In the present study we investigated the role of GATA-3 in the caudal raphe system, which is known to operate as a modulator of motor activity. We demonstrate that virtually all neurons in the caudal raphe nuclei that express GATA-3 also produce serotonin. Absence of GATA-3, as analyzed in chimeric -/- mice, affects the cytoarchitecture of serotonergic neurons in the caudal raphe nuclei. As a result the chimeras show a serious defect in their locomotor performance on a rotating rod. In sum, we conclude that GATA-3 plays a major role in the development of the serotonergic neurons of the caudal raphe nuclei, and that it is crucial for their role in locomotion.

Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear.

Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA-3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA-3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA-3 heterozygous and GATA-3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild-type littermates. This suggests that GATA-3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA-3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA-3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA-3 and the effects of null mutations on the ear suggest involvement of GATA-3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear.

GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice.

Human HDR (hypoparathyroidism, deafness and renal dysplasia)-syndrome is caused by haploinsufficiency of zinc-finger transcription factor GATA3. The hearing loss due to GATA3 haploinsufficiency has been shown to be peripheral in origin, but it is unclear to what extent potential aberrations in the outer hair cells (OHCs) contribute to this disorder. To further elucidate the pathophysiological mechanism underlying the hearing defect in HDR-syndrome, we investigated the OHCs in heterozygous Gata3-knockout mice at both the functional and morphological level. While the signal-to-noise ratios of distortion product otoacoustic emissions (DPOAE) in wild type mice did not change significantly during the first half-year of live, those in the heterozygous Gata3 mice decreased dramatically. In addition, both light microscopic and transmission electron microscopic analyses showed that the number of OHCs containing vacuoles was increased in the mutants. Together, these findings indicate that outer hair cell malfunctioning plays a major role in the hearing loss in HDR-syndrome.

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse beta-globin gene clusters.

By sequencing regions flanking the beta-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the beta-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5' sequences of mRNA from olfactory epithelium encoding beta-globin-proximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a "TATA" box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of beta-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous beta-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.

A mouse model for the cystic fibrosis delta F508 mutation.

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block.

CFTR expression and mucin secretion in cultured mouse gallbladder epithelial cells.

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in humans is frequently associated with progressive liver disease, which appears to result from obstruction of biliary ducts with mucous material. CFTR in the liver is expressed in the biliary epithelium. With the use of a mouse model for cystic fibrosis (CF) we have studied the relationship between CFTR expression and glycoprotein secretion in primary culture of mouse gallbladder epithelial cells (MGBC) MGBC in culture maintain a well-differentiated phenotype as shown by microscopy. The cells produce CFTR mRNA to levels comparable to the intact tissue. With patch-clamp analysis we could frequently observe a linear protein kinase A-regulated Cl- channel that shows all the major characteristics of human CFTR, although its conductance is lower (5 pS compared with 8 pS). MGBC in culture produce and secrete high molecular weight glycoproteins (HMG) in a time-dependent and temperature-sensitive manner. Secretion of HMG was not stimulated significantly by either adenosine 3',5'-cyclic monophosphate (cAMP), Ca2+, or protein kinase C agonists in this system. High concentrations (3 mM) of extracellular ATP stimulated secretion threefold, but low concentrations (0.3 mM) had no effect. Approximately one-third of the HMG produced and secreted consisted of mucin. Cultured MGBC from CFTR-deficient mice produced and secreted mucin to a similar extent as normal cells. We conclude that cultured mouse gallbladder cells are a convenient model to study both CFTR function and mucin secretion. In this system, we found no evidence for a direct link between mucin secretion and CFTR activity, as has been suggested for other cell types.

Cystic fibrosis transmembrane conductance regulator mediates the cyclic adenosine monophosphate-induced fluid secretion but not the inhibition of resorption in mouse gallbladder epithelium.

We have studied the physiological role of the cystic fibrosis (CF) gene product (cystic fibrosis transmembrane conductance regulator [CFTR]) in gallbladder epithelium using a knockout mouse model for CF. We found that normal mouse gallbladder epithelium expresses functional CFTR as shown by reverse-transcription polymerase chain reaction (RT-PCR) analysis and Ussing chamber experiments. Gallbladders from Cftr -/- mice were structurally intact as shown by microscopic and physiological parameters but lacked the cyclic adenosine monophosphate (cAMP)-induced chloride current observed in normal gallbladders. In fluid transport measurements, normal and Cftr -/- gallbladders were equally active in basal resorption. The addition of forskolin, which activates CFTR anion channel activity through the cAMP system, resulted in net fluid secretion in normal gallbladders. In contrast, Cftr -/- gallbladders were unable to secrete fluid while a complete inhibition of resorption by forskolin was observed. We conclude that, in normal mouse gallbladder epithelium, cAMP-induced fluid secretion involves simultaneous inhibition of apical sodium chloride resorption and activation of CFTR. Our data support the hypothesis that gallbladder disease in CF is at least in part caused by a deficient secretory response to the endogenous cAMP-linked hormones VIP and secretin.

Conservation of sequence and structure flanking the mouse and human beta-globin loci: the beta-globin genes are embedded within an array of odorant receptor genes.

In mouse and human, the beta-globin genes reside in a linear array that is associated with a positive regulatory element located 5' to the genes known as the locus control region (LCR). The sequences of the mouse and human beta-globin LCRs are homologous, indicating conservation of an essential function in beta-globin gene regulation. We have sequenced regions flanking the beta-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of approximately 40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human beta-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3' of the beta-globin locus in chicken. The data demonstrate an evolutionarily conserved genomic organization for the beta-globin locus and suggest a possible role for the beta-globin LCR in control of expression of these odorant receptor genes and/or the presence of mechanisms to separate regulatory signals in different tissues.

The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4.

In this paper, we show that the transcription factor GATA3 is dynamically expressed during hindbrain development. Function of GATA3 in ventral rhombomere (r) 4 is dependent on functional GATA2, which in turn is under the control of Hoxb1. In particular, the absence of Hoxb1 results in the loss of GATA2 expression in r4 and the absence of GATA2 results in the loss of GATA3 expression. The lack of GATA3 expression in r4 inhibits the projection of contralateral vestibuloacoustic efferent neurons and the migration of facial branchiomotor neurons similar to Hoxb1-deficient mice. Ubiquitous expression of Hoxb1 in the hindbrain induces ectopic expression of GATA2 and GATA3 in ventral r2 and r3. These findings demonstrate that GATA2 and GATA3 lie downstream of Hoxb1 and provide the first example of Hox pathway transcription factors within a defined population of vertebrate motor neurons.