SUMMARYThe inner cell mass (ICM) of the implanting mammalian blastocyst comprises two lineages: the pluripotent epiblast (EPI) and primitive endoderm (PrE). We have identified platelet-derived growth factor receptor alpha (PDGFR) as an early marker of the PrE lineage and its derivatives in both mouse embryos and ex vivo paradigms of extra-embryonic endoderm (ExEn). By combining live imaging of embryos and embryo-derived stem cells expressing a histone H2B-GFP fusion reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we found that Pdgfra expression coincides with that of GATA6, the earliest expressed transcriptional regulator of the PrE lineage. We show that GATA6 is required for the activation of Pdgfra expression. Using pharmacological inhibition and genetic inactivation we addressed the role of the PDGF pathway in the PrE lineage. Our results demonstrate that PDGF signaling is essential for the establishment, and plays a role in the proliferation, of XEN cells, which are isolated from mouse blastocyst stage embryos and represent the PrE lineage. Implanting Pdgfra mutant blastocysts exhibited a reduced number of PrE cells, an effect that was exacerbated by delaying implantation. Surprisingly, we also noted an increase in the number of EPI cells in implantation-delayed Pdgfra-null mutants. Taken together, our data suggest a role for PDGF signaling in the expansion of the ExEn lineage. Our observations also uncover a possible role for the PrE in regulating the size of the pluripotent EPI compartment.

Human centronuclear and myotubular myopathies belong to a genetically heterogeneous nosological group with clinical variability ranging from fatal disorder to mild weakness. The severe X-linked form is attributed to more than 200 different mutations in the myotubularin encoding gene (MTM1). In contrast, there are no reports regarding the molecular etiology or linkage studies on the autosomal forms of the disease. Labrador retrievers affected by spontaneous centronuclear myopathy (cnm) have clinical and histological features of the human disorder and represent the first model of recessive autosomal centronuclear myopathy. We previously mapped the cnm locus to the centromeric region of canine chromosome 2. No gene of the MTM1 family maps to the human homologous chromosomal region. Described herein is a disease-associated insertion within PTPLA exon 2, found in both alleles of all affected Labradors and in a single allele in obligate carriers. The inserted tRNA-derived short interspersed repeat element (SINE) has a striking effect on the maturation of PTPLA mRNA, whereby it can be spliced out, partially exonized or involved in multiple exon-skipping. As a result, the amount of wild-type transcripts falls to 1% in affected muscles. This example therefore recapitulates cumulative SINE-associated transcriptional defects that have been previously described as exclusive consequences of independent mutations. Although the function of PTPLA in metazoa remains unknown, the characterization of a hypomorphic mutation in Labradors with centronuclear myopathy provides new clues about the molecular complexity of skeletal myofiber homeostasis. These results also suggest that impaired PTPLA signaling might be implicated in human myopathies.

The suppression levels induced by gentamicin on premature stop codons, caused by primary nonsense mutations found in muscular dystrophy patients, were assessed using a very sensitive dual reporter gene assay. Results show that: (i) the effect of gentamicin on readthrough is similar in cultured cells and in vivo in murine skeletal muscle; (ii) a wide variability of readthrough efficiency is obtained, depending on the mutation tested; (iii) due to the complexity of readthrough regulation, efficiency cannot be predicted by the nucleotide context of the stop codon; (iv) only a minority of premature stop codons found in patients show a significant level of readthrough, and would thus be amenable to this pharmacological treatment, given our present understanding of the problem. These results probably provide an explanation for the relative failure of clinical trials reported to date using gentamicin to treat diseases due to premature stop codons, and emphasize that preliminary assays in cell culture provide valuable information concerning the potential efficiency of pharmacological treatments.

In the mouse, more than 16 loci are associated with mutant phenotypes that include defective pigmentation, aberrant targeting of lysosomal enzymes, prolonged bleeding, and immunodeficiency, the result of defective biogenesis of cytoplasmic organelles: melanosomes, lysosomes, and various storage granules. Many of these mouse mutants are homologous to the human HermanskyPudlak syndrome (HPS), Chediak-Higashi syndrome, and Griscelli syndrome. We have mapped and positionally cloned one of these mouse loci, buff (bf), which has a mutant phenotype similar to that of human HPS. Mouse bf results from a mutation in Vps33a and thus is homologous to the yeast vacuolar protein-sorting mutant vps33 and Drosophila carnation (car). This is the first found defect of the class C vacuole͞prevacuole-associated target soluble Nethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) complex in mammals and the first mammalian mutant found that is directly homologous to a vps mutation of yeast. VPS33A thus is a good candidate gene for a previously uncharacterized form of human HPS.H ermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis in which oculocutaneous albinism, bleeding, and in most cases pulmonary fibrosis result from defects of melanosomes, platelet-dense granules, and lysosomes (1-4). Somewhat similar disorders, Chediak-Higashi and Griscelli syndromes, are additionally associated with severe immunodeficiency (2, 3). Important clues to the pathogenesis of these disorders have come from the mouse, in which Ͼ16 loci have been associated with mutant phenotypes similar to those of human HPS, Chediak-Higashi syndrome, and Griscelli syndrome (5, 6). Several of these genes have been identified recently and in a number of cases have been shown to result in homologous disorders in mice and humans (2-4). Although the functions of many of the corresponding gene products remain unknown, several are involved in various aspects of trafficking proteins to nascent organelles, particularly melanosomes, lysosomes, and cytoplasmic granules. In the yeast, Ͼ65 proteins have been implicated in biogenesis of the cytoplasmic vacuole, including the products of Ͼ40 vacuolar protein-sorting (vps) loci required for trafficking newly synthesized proteins from the late Golgi͞trans-Golgi network to the vacuole (7, 8). It seems likely that at least as many proteins are associated with organellar biogenesis in mammals.We have mapped and positionally cloned the mouse buff (bf ) locus, which is characterized by recessive coat-color hypopigmentation and mild platelet-storage pool deficiency but has little if any effect on lysosomal function. We find that mouse bf results from a missense substitution in Vps33a, a homologue of yeast vps33. The bf mutation results in defective melanosome morphology and melanogenesis both in vivo and in vitro. Expression of wild-type Vps33a in transfected mouse bf-mutant melanocytes complements this aberrant phenotype, whereas expression of bf-mutant Vps33a does not. These results establish murine bf a...