Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and ß-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

Deficiency of the cytoskeletal protein SPECC1L leads to oblique facial clefting.

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

Mutations in the RNA granule component TDRD7 cause cataract and glaucoma.

The precise transcriptional regulation of gene expression is essential for vertebrate development, but the role of posttranscriptional regulatory mechanisms is less clear. Cytoplasmic RNA granules (RGs) function in the posttranscriptional control of gene expression, but the extent of RG involvement in organogenesis is unknown. We describe two human cases of pediatric cataract with loss-of-function mutations in TDRD7 and demonstrate that Tdrd7 nullizygosity in mouse causes cataracts, as well as glaucoma and an arrest in spermatogenesis. TDRD7 is a Tudor domain RNA binding protein that is expressed in lens fiber cells in distinct TDRD7-RGs that interact with STAU1-ribonucleoproteins (RNPs). TDRD7 coimmunoprecipitates with specific lens messenger RNAs (mRNAs) and is required for the posttranscriptional control of mRNAs that are critical to normal lens development and to RG function. These findings demonstrate a role for RGs in vertebrate organogenesis.

Transfusion independence and HMGA2 activation after gene therapy of human ß-thalassaemia.

The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

Apc inhibition of Wnt signaling regulates supernumerary tooth formation during embryogenesis and throughout adulthood.

The ablation of Apc function or the constitutive activation of beta-catenin in embryonic mouse oral epithelium results in supernumerary tooth formation, but the underlying mechanisms and whether adult tissues retain this potential are unknown. Here we show that supernumerary teeth can form from multiple regions of the jaw and that they are properly mineralized, vascularized, innervated and can start to form roots. Even adult dental tissues can form new teeth in response to either epithelial Apc loss-of-function or beta-catenin activation, and the effect of Apc deficiency is mediated by beta-catenin. The formation of supernumerary teeth via Apc loss-of-function is non-cell-autonomous. A small number of Apc-deficient cells is sufficient to induce surrounding wild-type epithelial and mesenchymal cells to participate in the formation of new teeth. Strikingly, Msx1, which is necessary for endogenous tooth development, is dispensable for supernumerary tooth formation. In addition, we identify Fgf8, a known tooth initiation marker, as a direct target of Wnt/beta-catenin signaling. These studies identify key mechanistic features responsible for supernumerary tooth formation.