A SAGE approach to identifying novel trans-acting factors involved in the X inactivation process.

X chromosome inactivation ensures the dosage compensation of X-linked genes in XX females compared to their XY male counterpart. It is characterised by the specific recruitment of an inhibitory ribonucleoprotein complex involving the non-coding Xist RNA to the presumptive inactive X chromosome and associated chromatin modifications, which result in the transcriptional silencing of the X chromosome. As an approach to the identification of some of the potential molecular players in this process we have performed comparative transcriptional profiling of mouse 6.5-dpc (days post-coitum) female and male embryos using a modified SAGE (Serial analysis of gene expression) technique which allows the analysis of small quantities of biological material. At 6.5 dpc, a moment when random X inactivation of embryonic tissues has just been achieved, some two hundred transcripts that were significantly enriched in the female gastrula compared to its male counterpart could be identified. The validation of an association with the X inactivation process of a subset of these transcripts has been studied, ex vivo, in differentiating female and male ES cells and in female ES cells in which the establishment of X inactivation is interrupted through the post-transcriptional inhibition of Xist synthesis.

Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling.

Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). We have analyzed the molecular mechanism by which Tsg regulates BMP signaling. Overexpression of Tsg mRNA in Xenopus embryos has ventralizing effects similar to Xolloid, a metalloprotease that cleaves Chordin. In embryos dorsalized by LiCl treatment, microinjection of Xolloid or Tsg mRNA restores the formation of trunk-tail structures, indicating an increase in BMP signaling. Microinjection of Tsg mRNA leads to the degradation of endogenous Chordin fragments generated by Xolloid. The ventralizing activities of Tsg require an endogenous Xolloid-like activity, as they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay revealed that Tsg has two distinct and sequential activities on BMP signaling. First, Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. Second, after cleavage of Chordin by Xolloid, Tsg competes the residual anti-BMP activity of Chordin fragments and facilitates their degradation. This molecular pathway, in which Xolloid switches the activity of Tsg from a BMP antagonist to a pro-BMP signal once all endogenous full-length Chordin is degraded, may help explain how sharp borders between embryonic territories are generated.

Identification and expression of the mammalian homologue of Bicaudal-C.

Translational activation and repression play an important role in the spatial-temporal regulation of gene expression in embryonic development. Bicaudal-C is an RNA-binding molecule believed to function at this post-transcriptional level. Loss-of-function mutants in Drosophila affect anterior-posterior patterning because of ectopic and premature translation of the posterior determinant oskar. The Xenopus homologue of Bicaudal-C is one of the few molecules that, when microinjected ectopically, results in endoderm formation in the absence of mesoderm induction. Here we report the sequence and expression pattern of the murine and human homologues of Bicaudal-C. The human gene is located on chromosome 10q21.2. Expression analysis in mouse using in situ hybridization detects expression of Bicaudal-C not only in domains detected in Xenopus, but also in previously unreported regions. As in Xenopus, mouse Bicaudal-C mRNA is found in the growing oocyte, Hensen's node, and the developing kidney. Additionally, at later stages it is strongly expressed in the developing gut endoderm, in areas of cartilage formation, in pleuro-peritoneal membrane derivatives, in lung mesenchyme, and in the stroma of the ovary. We conclude that mouse Bicaudal-C is a maternally provided gene product that is tightly regulated during mammalian cell differentiation.

Otx2 is required for visceral endoderm movement and for the restriction of posterior signals in the epiblast of the mouse embryo.

Genetic and embryological experiments have demonstrated an essential role for the visceral endoderm in the formation of the forebrain; however, the precise molecular and cellular mechanisms of this requirement are poorly understood. We have performed lineage tracing in combination with molecular marker studies to follow morphogenetic movements and cell fates before and during gastrulation in embryos mutant for the homeobox gene Otx2. Our results show, first, that Otx2 is not required for proliferation of the visceral endoderm, but is essential for anteriorly directed morphogenetic movement. Second, molecules that are normally expressed in the anterior visceral endoderm, such as Lefty1 and Mdkk1, are not expressed in Otx2 mutants. These secreted proteins have been reported to antagonise, respectively, the activities of Nodal and Wnt signals, which have a role in regulating primitive streak formation. The visceral endoderm defects of the Otx2 mutants are associated with abnormal expression of primitive streak markers in the epiblast, suggesting that anterior epiblast cells acquire primitive streak characteristics. Taken together, our data support a model whereby Otx2 functions in the anterior visceral endoderm to influence the ability of the adjacent epiblast cells to differentiate into anterior neurectoderm, indirectly, by preventing them from coming under the influence of posterior signals that regulate primitive streak formation.

Gene expression profiles in normal and Otx2-/- early gastrulating mouse embryos.

The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and Otx2(-/-) 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation.

The early expression of Rev-erbbeta occurs in the developing nervous system of mouse embryo.

The orphan ligand nuclear receptor Rev-erbbeta acts in vitro as a negative regulator of transcription. However, its precise physiological role is still unknown. As a first attempt to better understand its biological function, we have studied the distribution and the localization of the Rev-erbbeta mRNA transcripts in different mouse embryonal carcinoma cell lines, in mouse embryos and adult tissues. Our results indicated that Rev-erbbeta transcripts are present in both the non-differentiated and differentiated F9 cells into either parietal or visceral endoderm. At 12.5 days of gestation (E12.5) of mouse embryos, Rev-erbbeta transcripts were localized only in the developing nervous system. In contrast, at later stages of development as well as in the adult, its messengers were widely distributed. These results suggest that Rev-erbbeta may have different roles at the different stages of mouse development, with a more specific role in the nervous system at earlier stages.

Structure and expression of Wnt13, a novel mouse Wnt2 related gene.

We have identified a novel mouse member of the Wnt family, Wnt13. Among mouse Wnt genes, Wnt13 is most closely related to Wnt2. Sequence comparisons and chromosomal localization strongly suggest that Wnt13, rather than Wnt2, is the mouse orthologue of both the human WNT13 and Xenopus XWnt2 genes. Wnt13 is expressed in the embryonic mesoderm during gastrulation. At later stages, transcripts are detected in the dorsal midline of the diencephalon and mesencephalon, the heart primordia, the periphery of the lung bud and the otic and optic vesicles. These data suggest that Wnt13 function might partially overlap with those of other Wnt genes in the cell signaling mechanisms controlling mesoderm specification during gastrulation and some aspects of brain, heart and lung formation.