Malcolm Fisher

Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06–1.23 Mb upstream of SOX9, and microdeletions both ∼1.5 Mb centromeric and ∼1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

Endogenous Retrotransposition Activates Oncogenic Pathways in Hepatocellular Carcinoma

Summary LINE-1 (L1) retrotransposons are mobile genetic elements comprising ∼17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic β-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2−/− mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.

EMAGE mouse embryo spatial gene expression database: 2010 update

EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (∼19 000 gene) ‘EURExpress’ dataset into EMAGE.

Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.

Integrating technologies for comparing 3D gene expression domains in the developing chick limb

Chick embryos are good models for vertebrate development due to their accessibility and manipulability. Recent large increases in available genomic data from both whole genome sequencing and EST projects provide opportunities for identifying many new developmentally important chicken genes. Traditional methods of documenting when and where specific genes are expressed in embryos using wholemount and section in-situ hybridisation do not readily allow appreciation of 3-dimensional (3D) patterns of expression, but this can be accomplished by the recently developed microscopy technique, Optical Projection Tomography (OPT). Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared. Novel protocols are used to compare 3D expression domains of 7 genes known to be involved in chick wing development. This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression. Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

The associations between age and the development of laboratory abnormalities and treatment discontinuation for reasons other than virological failure in the first year of highly active antiretroviral therapy

The aim of this study was to describe the relationship between age and the time to treatment discontinuation in the absence of virological failure as well as the development of specific laboratory abnormalities, in patients starting highly active antiretroviral therapy (HAART) for the first time.

Esrrg functions in early branch generation of the ureteric bud and is essential for normal development of the renal papilla

Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5–16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg−/−) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT.

Identification of genes downstream of the Shh signalling in the developing chick wing and syn-expressed with Hoxd13 using microarray and 3D computational analysis

Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation.

Comparative Analysis of 3D Expression Patterns of Transcription Factor Genes and Digit Fate Maps in the Developing Chick Wing

Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.

3D expression patterns of cell cycle genes in the developing chick wing and comparison with expression patterns of genes implicated in digit specification

Sonic hedgehog (Shh) signalling controls integrated specification of digit pattern and growth in the chick wing but downstream gene networks remain to be unravelled. We analysed 3D expression patterns of genes encoding cell cycle regulators using Optical Projection Tomography. Hierarchical clustering of spatial matrices of gene expression revealed a dorsal layer of the wing bud, in which almost all genes were expressed, and that genes encoding positive cell cycle regulators had similar expression patterns while those of N‐myc and CyclinD2 were distinct but closely related. We compared these patterns computationally with those of genes implicated in digit specification and Ptch1, 50 genes in total. Nineteen genes have similar posterior expression to Ptch1, including Hoxd13, Sall1, Hoxd11, and Bmp2, all likely Gli targets in mouse limb, and cell cycle genes, N‐myc, CyclinD2. We suggest that these genes contribute to a network integrating digit specification and growth in response to Shh. Developmental Dynamics 240:1278–1288, 2011.