Role for the pleckstrin homology domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase-regulated muscle differentiation.

In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.

Molecular dissection reveals decreased activity and not dominant negative effect in human OTX2 mutants.

The paired-type homeodomain transcription factor Otx2 is essential for forebrain and eye development. Severe ocular malformations in humans have recently been associated with heterozygous OTX2 mutations. To document the molecular defects in human mutants, Otx2 structural characterization was carried out. A collection of deletion and point mutants was created to perform transactivation, DNA binding, and subcellular localization analyses. Transactivation was ascribed to both N- and C-termini of the protein, and DNA binding to the minimal homeodomain, where critical amino acid residues were identified. Acute nuclear localization appeared controlled by a nuclear localization sequence located within the homeodomain which acts in conjunction with a novel nuclear retention domain that we unraveled located in the central part of the protein. This region, which is poorly conserved among Otx proteins, was also endowed with dominant negative activity suggesting that it might confer unique properties to Otx2. Molecular diagnostic of human mutant OTX2 proteins discriminates hypomorphic and loss of function mutations from other mutations that may not be relevant to ocular pathology.

Temporal and spatial delineation of mouse Otx2 functions by conditional self-knockout.

To identify the independent spatial and temporal activities of the essential developmental gene the Otx2, the germline mutation of which is lethal at embryonic day 8.5, we floxed one allele and substituted the other with an inducible CreER recombinase gene. This makes 'trans' self-knockout possible at any developmental stage. The transient action of tamoxifen pulses allows time-course mutation. We demonstrate efficient temporal knockout and demarcate spatio-temporal windows in which Otx2 controls the head, brain structures and body development.

Alternative usage of Otx2 promoters during mouse development.

Our previous structural analysis of mouse Otx2 transcripts has revealed the existence of three different promoters and suggested that the corresponding mRNAs could exhibit specific expression patterns. Here, we analyze the precise dynamics of their expression throughout mouse development. Their spatial distribution was determined by isoform-specific in situ hybridization and their relative abundance by real-time reverse transcriptase-polymerase chain reaction. Although the three promoters may be used in the same areas, we show that transcription preferentially occurs from the proximal promoter at onset of gene activity in early embryogenesis, and switches to the more distal one in most of the sites of expression in the adult brain. During gestation, their relative utilization becomes inverted. The third promoter, which shows no activity in embryonic stem cells and is moderately expressed during embryogenesis, is mostly used in specific areas derived from the rostral part of the neural tube.

New Otx2 mRNA isoforms expressed in the mouse brain.

The mouse Otx2 gene is essential throughout head and brain development, from anterior-posterior polarity determination and neuroectoderm induction to post-natal sensory organ maturation. These numerous activities must rely on a very finely tuned regulation of expression. In order to understand the molecular control of the Otx2 gene, we set out to isolate its promoter. During this quest, we identified three remote transcription start sites, two defining two new upstream exons and one mapping within the previously reported first exon. The three transcripts differed in their 5' non-coding region but encoded the same protein. The transcription start nucleotides of each mRNA species have been mapped by RNase protection assays and by an RNA circularization technique. We have demonstrated that they are all used and linked to functional promoters. In addition to leader versatility, we also detected alternative splicing within the coding sequence that gives rise to a new protein endowed with an 8 amino-acid insertion upstream of the homeodomain. Combined analysis of the relative abundance of Otx2 mRNA isoforms in representative tissues and in situ hybridization studies revealed distinct spatial and temporal, although partially overlapping, expression patterns of the mRNA isoforms. These findings provide new clues to a better understanding of the relationships between Otx2 gene architecture and its complex regulatory requirements.