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DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Using chromatin immunoprecipitation combined with genomic microarrays we have identified targets of No tail (Ntl), a zebrafish Brachyury ortholog that plays a central role in mesoderm formation. We show that Ntl regulates a downstream network of other transcription factors and identify an in vivo Ntl binding site that resembles the consensus T-box binding site (TBS) previously identified by in vitro studies. We show that the notochord-expressed gene floating head ( flh ) is a direct transcriptional target of Ntl and that a combination of TBSs in the flh upstream region are required for Ntl-directed expression. Using our genome-scale data we have assembled a preliminary gene regulatory network that begins to describe mesoderm formation and patterning in the early zebrafish embryo.

Members of the REEP (Receptor expression enhancing protein) family contain a TB2/DP1, HVA22 domain that is involved in intracellular trafficking and secretion. Consistent with the presence of this domain, REEP1 and REEP3 enhance the expression of odorant and taste receptors in mammals, while mutation of these genes causes defects in neural development. REEP4 was identified in the course of a functional antisense morpholino oligonucleotide screen searching for genes involved in the early development of Xenopus tropicalis: although over-expression of the gene causes no phenotype, embryos lacking REEP4 develop a slightly kinked body axis and are paralysed. At tailbud stages of development, REEP4 is expressed in the somites and neural tube. The paralysis observed in embryos lacking REEP4 might therefore be caused by defects in the nervous system or in muscle. To address this point, we examined the expression of various neural and muscle markers and found that although all are expressed normally at early stages of development, many are down regulated by the tailbud stage. This suggests that REEP4 plays a role in the maintenance of both the nervous system and the musculature.

The Xenopus ectoderm consists of two populations of cells, superficial polarised epithelial cells and deep, non-epithelial cells. These two cell types differ in their developmental fate. In the neural ectoderm, primary neurons are derived only from the deep cells. In the epidermal ectoderm, superficial cells express high levels of differentiation markers, while most of the deep cells do not differentiate until later when they produce the stratified adult epidermis. However, few molecular differences are known between the deep and superficial cells. Here, we have undertaken a systematic approach to identify genes that show layer-restricted expression by microarray analysis of deep and superficial cells at the gastrula stage, followed by wholemount in situ hybridisation. We have identified 32 differentially expressed genes, of which 26 show higher expression in the superficial layer and 6 in the deep layer and describe their expression at the gastrula and neurula stage. One of the identified genes is the transcription factor Grhl3, which we found to be expressed in the superficial layer of the gastrula ectoderm and the neurula epidermis. By using markers identified in this work, we show that Grlh3 promotes superficial gene expression in the deep layer of the epidermis. Concomitantly, deep layer specific genes are switched off, showing that Grlh3 can promote deep cells to take on a superficial cell identity in the embryonic epidermis.

We have generated an improved assembly and gene annotation of the pig X chromosome, and a first draft assembly of the pig Y chromosome, by sequencing BAC and fosmid clones, and incorporating information from optical mapping and fibre-FISH. The X chromosome carries 1,014 annotated genes, 689 of which are protein-coding. Gene order closely matches that found in Primates (including humans) and Carnivores (including cats and dogs), which is inferred to be ancestral. Nevertheless, several protein-coding genes present on the human X chromosome were absent from the pig (e.g. the cancer/testis antigen family) or inactive (e.g. AWAT1), and 38 pig-specific X-chromosomal genes were annotated, 22 of which were olfactory receptors. The pig Y chromosome assembly focussed on two clusters of male-specific low-copy number genes, separated by an ampliconic region including the HSFY gene family, which together make up most of the short arm. Both clusters contain palindromes with high sequence identity, presumably maintained by gene conversion. The long arm of the chromosome is almost entirely repetitive, containing previously characterised sequences. Many of the ancestral X-related genes previously reported in at least one mammalian Y chromosome are represented either as active genes or partial sequences. This sequencing project has allowed us to identify genes - both single copy and amplified - on the pig Y, to compare the pig X and Y chromosomes for homologous sequences, and thereby to reveal mechanisms underlying pig X and Y chromosome evolution.