Unr defines a novel class of nucleoplasmic reticulum involved in mRNA translation.

Unr (officially known as CSDE1) is a cytoplasmic RNA-binding protein with roles in the regulation of mRNA stability and translation. In this study, we identified a novel function for Unr, which acts as a positive regulator of placental development. Unr expression studies in the developing placenta revealed the presence of Unr-rich foci that are apparently located in the nuclei of trophoblast giant cells (TGCs). We determined that what we initially thought to be foci, were actually cross sections of a network of double-wall nuclear membrane invaginations that contain a cytoplasmic core related to the nucleoplasmic reticulum (NR). We named them, accordingly, Unr-NRs. Unr-NRs constitute a novel type of NR because they contain high levels of poly(A) RNA and translation factors, and are sites of active translation. In murine tissues, Unr-NRs are only found in two polyploid cell types, in TGCs and hepatocytes. In vitro, their formation is linked to stress and polyploidy because, in three cancer cell lines, cytotoxic drugs that are known to promote polyploidization induce their formation. Finally, we show that Unr is required in vivo for the formation of Unr-containing NRs because these structures are absent in Unr-null TGCs.

Conditional inactivation of the E-cadherin gene in thyroid follicular cells affects gland development but does not impair junction formation.

We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.

E-cadherin controls adherens junctions in the epidermis and the renewal of hair follicles.

E-cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E-cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E-cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and beta-catenin levels were not strongly altered in the mutant skin. We conclude that E-cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E-cadherin.

Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus.

Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (-/+) or both (-/-) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr(+/+), unr(+/-), and unr(-/-) cell lines. Translation directed by the HRV IRES was severely impaired in unr(-/-) cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr(-/-) cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

E-cadherin is required for the correct formation of autotypic adherens junctions of the outer mesaxon but not for the integrity of myelinated fibers of peripheral nerves.

The calcium-dependent adhesion protein E-cadherin is present in noncompacted regions of myelin sheaths in the peripheral nervous system. There, it is localized to electron-dense structures between membranes of the same Schwann cell referred to as autotypic adherens junctions. It has been suggested that the failure of E-cadherin-mediated adhesion might cause demyelination that proceeds in certain pathological states. To test the requirement of E-cadherin in peripheral nerves, we used tissue-specific gene ablation techniques based on the Cre/LoxP system. We show that E-cadherin deficiency does not cause significant demyelination up to the age of 15 months. Immunostainings for nodal sodium channels, the paranodal protein Caspr1, and the juxtaparanodal potassium channels Kv1.1 and Kv1.2 revealed that E-cadherin is not necessary to maintain the general functional architecture of the nodal region. On the ultrastructural level, we detected a widening of the outer mesaxon accompanied by a loss of electron-dense cytoplasmic areas. We conclude that E-cadherin is required for the proper establishment and/or the maintenance of the outer mesaxon in myelinated PNS fibers but is dispensable for proper nerve function.

E-cadherin is a survival factor for the lactating mouse mammary gland.

The Ca(++)-dependent cell adhesion molecule E-cadherin is expressed throughout mouse development and in adult tissues. Classical gene targeting has demonstrated that E-cadherin-deficient embryos die at the blastocyst stage. To study the involvement of E-cadherin in organogenesis, a conditional gene inactivation scheme was undertaken using the bacteriophage P1 recombinase Cre/loxP system. Mice with homozygous loxP sites in both alleles of the E-cadherin (Cdh1) gene were generated and these mice were crossed with transgenic mice with the Cre recombinase under the control of the hormone-inducible MMTV promoter. This resulted in deletion of the E-cadherin gene in the differentiating alveolar epithelial cells of the mammary gland. The mutant mammary gland developed normally up to 16-18 days of pregnancy but exhibited a dramatic phenotype around parturition. The production of milk proteins was so drastically reduced that adult mutant mothers could not suckle their offspring. Thus, the lack of E-cadherin affected the terminal differentiation program of the lactating mammary gland. In concordance with this finding, the prolactin-dependent activation of the transcription factor Stat5a was initiated but not maintained in the mutant gland. Instead, without E-cadherin massive cell death was observed at parturition and the mutant mammary gland at this stage resembled that of the involuted gland normally seen after weaning. These results demonstrate an essential role for E-cadherin in the function of differentiated alveolar epithelial cells. No tumors were detected in mutant glands lacking E-cadherin.