A 3D searchable database of transgenic zebrafish gal4 and cre lines for functional neuroanatomy studies

Citation: Marquart, G. D., Tabor, K. M., Brown, M., Strykowski, J. L., Varshney, G. K., LaFave, M. C., . . . Burgess, H. A. (2015). A 3D searchable database of transgenic zebrafish gal4 and cre lines for functional neuroanatomy studies. Frontiers in Neural Circuits, 9(November), 1-17. doi:10.3389/fncir.2015.00078Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest (ROI) or neuroanatomical area. This software also enables the intersectional expression of transgenes to be predicted, a feature which we validated by detecting cells with co-expression of Cre and Gal4. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle, and skin through the incorporation of microRNA binding sites in a synthetic 3? untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70% of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish. © 2015 Marquart, Tabor, Brown, Strykowski, Varshney, LaFave, Mueller, Burgess, Higashijima and Burgess

Glycosylation of Twisted Gastrulation is Required for BMP Binding and Activity during Craniofacial Development

Twisted gastrulation (TWSG1) is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs) in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1) is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to glycosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action

Integrated inference and evaluation of host-fungi interaction networks

Fungal microorganisms frequently lead to life-threatening infections. Within this group of pathogens, the commensal Candida albicans and the filamentous fungus Aspergillus fumigatus are by far the most important causes of invasive mycoses in Europe. A key capability for host invasion and immune response evasion are specific molecular interactions between the fungal pathogen and its human host. Experimentally validated knowledge about these crucial interactions is rare in literature and even specialized host pathogen databases mainly focus on bacterial and viral interactions whereas information on fungi is still sparse. To establish large-scale host fungi interaction networks on a systems biology scale, we develop an extended inference approach based on protein orthology and data on gene functions. Using human and yeast intraspecies networks as template, we derive a large network of pathogen host interactions (PHI). Rigorous filtering and refinement steps based on cellular localization and pathogenicity information of predicted interactors yield a primary scaffold of fungi human and fungi mouse interaction networks. Specific enrichment of known pathogenicity-relevant genes indicates the biological relevance of the predicted PHI. A detailed inspection of functionally relevant subnetworks reveals novel host fungal interaction candidates such as the Candida virulence factor PLB1 and the anti-fungal host protein APP. Our results demonstrate the applicability of interolog-based prediction methods for host fungi interactions and underline the importance of filtering and refinement steps to attain biologically more relevant interactions. This integrated network framework can serve as a basis for future analyses of high-throughput host fungi transcriptome and proteome data