Salla disease and infantile sialic acid storage disorder are autosomal recessive neurodegenerative diseases characterized by loss of a lysosomal sialic acid transport activity and the resultant accumulation of free sialic acid in lysosomes. Genetic analysis of these diseases has identified several unique mutations in a single gene encoding a protein designated sialin (Verheijen, F. W., Verbeek, E., Aula, N., Beerens, C. E., Havelaar, A. C., Joosse, M., Peltonen, L., Aula, P., Galjaard, H., van der Spek, P. J., and Mancini, G. M. (1999) Nat. Genet. 23, 462-465; Aula, N., Salomaki, P., Timonen, R., Verheijen, F., Mancini, G., Mansson, J. E., Aula, P., and Peltonen, L. (2000) Am. J. Hum. Genet. 67, 832-840). From the biochemical phenotype of the diseases and the predicted polytopic structure of the protein, it has been suggested that sialin functions as a lysosomal sialic acid transporter. Here we directly demonstrate that this activity is mediated by sialin and that the recombinant protein has functional characteristics similar to the native lysosomal sialic acid transport system. Furthermore, we describe the effect of disease-causing mutations on the protein. We find that the majority of the mutations are associated with a complete loss of activity, while the mutations associated with the milder forms of the disease lead to reduced, but residual, function. Thus, there is a direct correlation between sialin function and the disease state. In addition, we find with one mutation that the protein is retained in the endoplasmic reticulum, indicating that altered trafficking of sialin is also associated with disease. This analysis of the molecular mechanism of sialic acid storage disorders is a further step in identifying therapeutic approaches to these diseases.

CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F 0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F 0 generation. Over the last couple of years, CRISPR/Cas9 has revolutionized reverse genetic studies in non-mammalian vertebrate model organisms 1-3 , and has further empowered the use of Xenopus and zebrafish as model organisms for studying development and human disease 4-6. In particular, F 0 CRISPR/Cas9-mediated gene disruption in non-mammalian vertebrates has emerged as a cost-effective method to rapidly assign causality to genetic variants in candidate disease genes identified from human patient exome sequencing 7-11. This can assist clinical geneticists in providing timely genetic diagnosis and counseling to patients and affected families, thereby favoring societal and economic impact of findings. CRISPR/Cas9 mediated F 0 mosaic mutant embryos are also increasingly employed as an alternative to antisense morpholino oligomers (MOs) 12,13 to investigate gene function and genetic interactions in developing embryos 14 , thus expanding the toolbox for cell and developmental biologists. An important consideration in CRISPR/Cas9 mutational studies is identifying gRNAs that produce a high frequency of loss-of-function mutations in the appropriate coding exons and hence generate highly penetrant specific F 0 phenotypes 15. During gRNA design, considerations include the possibilities of reading frame preservation

Background Respiratory system development is regulated by a complex series of endoderm – mesoderm interactions that are not fully understood. Recently Xenopus has emerged as an alternative model to investigate early respiratory system development, but the extent to which the morphogenesis and molecular pathways involved are conserved between Xenopus and mammals has not been systematically documented. Results In this study we provide a histological and molecular atlas of Xenopus respiratory system development, focusing on Nkx2.1+ respiratory cell fate specification in the developing foregut. We document the expression patterns of Wnt/β-catenin, FGF, and BMP signaling components in the foregut and show that the molecular mechanisms of respiratory lineage induction are remarkably conserved between Xenopus and mice. Finally, using a number of functional experiments we refine the epistatic relationships between FGF, Wnt and BMP signaling in early Xenopus respiratory system development. Conclusions We demonstrate that Xenopus trachea and lung development, before metamorphosis, is comparable at the cellular and molecular levels to embryonic stages of mouse respiratory system development between E8.5 to E10.5. This molecular atlas provides a fundamental starting point for further studies using Xenopus as a model to define the conserved genetic programs controlling early respiratory system development.

Two species of the clawed frog family, Xenopus laevis and X. tropicalis , are widely used as tools to investigate both normal and disease-state biochemistry, genetics, cell biology, and developmental biology. To support both frog specialist and non-specialist scientists needing access to these models for their research, a number of centralized resources exist around the world. These include centers that hold live and frozen stocks of transgenic, inbred and mutant animals and centers that hold molecular resources. This infrastructure is supported by a model organism database. Here, we describe much of this infrastructure and encourage the community to make the best use of it and to guide the resource centers in developing new lines and libraries.