Single-cell sequencing shows mosaic aneuploidy in most human embryos.

Mammalian preimplantation embryos often contain chromosomal defects that arose in the first divisions after fertilization and affect a subpopulation of cells - an event known as mosaic aneuploidy. In this issue of the JCI, Chavli et al. report single-cell genomic sequencing data for rigorous evaluation of the incidence and degree of mosaic aneuploidy in healthy human in vitro fertilization (IVF) embryos. Remarkably, mosaic aneuploidy occurred in at least 80% of human blastocyst-stage embryos, with often less than 20% of cells showing defects. These findings confirm that mosaic aneuploidy is prevalent in human embryos, indicating that the process is a widespread event that rarely has clinical consequences. There are major implications for preimplantation genetic testing of aneuploidy (PGT-A), a test commonly used to screen and select IVF embryos for transfer. The application and benefit of this technology is controversial, and the findings provide more cause for caution on its use.

Molecular Biology of the WWOX Gene That Spans Chromosomal Fragile Site FRA16D.

It is now more than 20 years since the FRA16D common chromosomal fragile site was characterised and the WWOX gene spanning this site was identified. In this time, much information has been discovered about its contribution to disease; however, the normal biological role of WWOX is not yet clear. Experiments leading to the identification of the WWOX gene are recounted, revealing enigmatic relationships between the fragile site, its gene and the encoded protein. We also highlight research mainly using the genetically tractable model organism Drosophila melanogaster that has shed light on the integral role of WWOX in metabolism. In addition to this role, there are some particularly outstanding questions that remain regarding WWOX, its gene and its chromosomal location. This review, therefore, also aims to highlight two unanswered questions. Firstly, what is the biological relationship between the WWOX gene and the FRA16D common chromosomal fragile site that is located within one of its very large introns? Secondly, what is the actual substrate and product of the WWOX enzyme activity? It is likely that understanding the normal role of WWOX and its relationship to chromosomal fragility are necessary in order to understand how the perturbation of these normal roles results in disease.

Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 h u2787 Model of Fragile X Syndrome.

Zebrafish represent a valuable model for investigating the molecular and cellular basis of Fragile X syndrome (FXS). Reduced expression of the zebrafish FMR1 orthologous gene, fmr1, causes developmental and behavioural phenotypes related to FXS. Zebrafish homozygous for the hu2787 non-sense mutation allele of fmr1 are widely used to model FXS, although FXS-relevant phenotypes seen from morpholino antisense oligonucleotide (morpholino) suppression of fmr1 transcript translation were not observed when hu2787 was first described. The subsequent discovery of transcriptional adaptation (a form of genetic compensation), whereby mutations causing non-sense-mediated decay of transcripts can drive compensatory upregulation of homologous transcripts independent of protein feedback loops, suggested an explanation for the differences reported. We examined the whole-embryo transcriptome effects of homozygosity for fmr1 h u2787 at 2 days post fertilisation. We observed statistically significant changes in expression of a number of gene transcripts, but none from genes showing sequence homology to fmr1. Enrichment testing of differentially expressed genes implied effects on lysosome function and glycosphingolipid biosynthesis. The majority of the differentially expressed genes are located, like fmr1, on Chromosome 14. Quantitative PCR tests did not support that this was artefactual due to changes in relative chromosome abundance. Enrichment testing of the "leading edge" differentially expressed genes from Chromosome 14 revealed that their co-location on this chromosome may be associated with roles in brain development and function. The differential expression of functionally related genes due to mutation of fmr1, and located on the same chromosome as fmr1, is consistent with R.A. Fisher's assertion that the selective advantage of co-segregation of particular combinations of alleles of genes will favour, during evolution, chromosomal rearrangements that place them in linkage disequilibrium on the same chromosome. However, we cannot exclude that the apparent differential expression of genes on Chromosome 14 genes was, (if only in part), caused by differences between the expression of alleles of genes unrelated to the effects of the fmr1 h u2787 mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.

Non-self mutation: double-stranded RNA elicits antiviral pathogenic response in a Drosophila model of expanded CAG repeat neurodegenerative diseases.

Inflammation is activated prior to symptoms in neurodegenerative diseases, providing a plausible pathogenic mechanism. Indeed, genetic and pharmacological ablation studies in animal models of several neurodegenerative diseases demonstrate that inflammation is required for pathology. However, while there is growing evidence that inflammation-mediated pathology may be the common mechanism underlying neurodegenerative diseases, including those due to dominantly inherited expanded repeats, the proximal causal agent is unknown. Expanded CAG.CUG repeat double-stranded RNA causes inflammation-mediated pathology when expressed in Drosophila. Repeat dsRNA is recognized by Dicer-2 as a foreign or 'non-self' molecule triggering both antiviral RNA and RNAi pathways. Neither of the RNAi pathway cofactors R2D2 nor loquacious are necessary, indicating antiviral RNA activation. RNA modification enables avoidance of recognition as 'non-self' by the innate inflammatory surveillance system. Human ADAR1 edits RNA conferring 'self' status and when co-expressed with expanded CAG.CUG dsRNA in Drosophila the pathology is lost. Cricket Paralysis Virus protein CrPV-1A is a known antagonist of Argonaute-2 in Drosophila antiviral defense. CrPV-1A co-expression also rescues pathogenesis, confirming anti-viral-RNA response. Repeat expansion mutation therefore confers 'non-self' recognition of endogenous RNA, thereby providing a proximal, autoinflammatory trigger for expanded repeat neurodegenerative diseases.

Neurodegenerative diseases have genetic hallmarks of autoinflammatory disease.

The notion that one common pathogenic pathway could account for the various clinically distinguishable, typically late-onset neurodegenerative diseases might appear unlikely given the plethora of diverse primary causes of neurodegeneration. On the contrary, an autoinflammatory pathogenic mechanism allows diverse genetic and environmental factors to converge into a common chain of causality. Inflammation has long been known to correlate with neurodegeneration. Until recently this relationship was seen as one of consequence rather than cause-with inflammatory cells and events acting to 'clean up the mess' after neurological injury. This explanation is demonstrably inadequate and it is now clear that inflammation is at the very least, rate-limiting for neurodegeneration (and more likely, a principal underlying cause in most if not all neurodegenerative diseases), protective in its initial acute phase, but pernicious in its latter chronic phase.

Neuronal-specific impairment of heparan sulfate degradation in Drosophila reveals pathogenic mechanisms for Mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder resulting from the deficit of the N-sulfoglucosamine sulfohydrolase (SGSH) enzyme that leads to accumulation of partially-degraded heparan sulfate. MPS IIIA is characterized by severe neurological symptoms, clinically presenting as Sanfilippo syndrome, for which no effective therapy is available. The lysosomal SGSH enzyme is conserved in Drosophila and we have identified increased levels of heparan sulfate in flies with ubiquitous knockdown of SGSH/CG14291. Using neuronal specific knockdown of SGSH/CG14291 we have also observed a higher abundance of Lysotracker-positive puncta as well as increased expression of GFP tagged Ref(2)P supporting disruption to lysosomal function. We have also observed a progressive defect in climbing ability, a hallmark of neurological dysfunction. Genetic screens indicate proteins and pathways that can functionally modify the climbing phenotype, including autophagy-related proteins (Atg1 and Atg18), superoxide dismutase enzymes (Sod1 and Sod2) and heat shock protein (HSPA1). In addition, reducing heparan sulfate biosynthesis by knocking down sulfateless or slalom expression significantly worsens the phenotype; an important observation given that substrate inhibition is being evaluated clinically as a treatment for MPS IIIA. Identifying the cellular pathways that can modify MPS IIIA neuropathology is an essential step in the development of novel therapeutic approaches to prevent and/or ameliorate symptoms in children with Sanfilippo syndrome.

The Enemy within: Innate Surveillance-Mediated Cell Death, the Common Mechanism of Neurodegenerative Disease.

Neurodegenerative diseases comprise an array of progressive neurological disorders all characterized by the selective death of neurons in the central nervous system. Although, rare (familial) and common (sporadic) forms can occur for the same disease, it is unclear whether this reflects several distinct pathogenic pathways or the convergence of different causes into a common form of nerve cell death. Remarkably, neurodegenerative diseases are increasingly found to be accompanied by activation of the innate immune surveillance system normally associated with pathogen recognition and response. Innate surveillance is the cell's quality control system for the purpose of detecting such danger signals and responding in an appropriate manner. Innate surveillance is an "intelligent system," in that the manner of response is relevant to the magnitude and duration of the threat. If possible, the threat is dealt with within the cell in which it is detected, by degrading the danger signal(s) and restoring homeostasis. If this is not successful then an inflammatory response is instigated that is aimed at restricting the spread of the threat by elevating degradative pathways, sensitizing neighboring cells, and recruiting specialized cell types to the site. If the danger signal persists, then the ultimate response can include not only the programmed cell death of the original cell, but the contents of this dead cell can also bring about the death of adjacent sensitized cells. These responses are clearly aimed at destroying the ability of the detected pathogen to propagate and spread. Innate surveillance comprises intracellular, extracellular, non-cell autonomous and systemic processes. Recent studies have revealed how multiple steps in these processes involve proteins that, through their mutation, have been linked to many familial forms of neurodegenerative disease. This suggests that individuals harboring these mutations may have an amplified response to innate-mediated damage in neural tissues, and renders innate surveillance mediated cell death a plausible common pathogenic pathway responsible for neurodegenerative diseases, in both familial and sporadic forms. Here we have assembled evidence in favor of the hypothesis that neurodegenerative disease is the cumulative result of chronic activation of the innate surveillance pathway, triggered by endogenous or environmental danger or damage associated molecular patterns in a progressively expanding cascade of inflammation, tissue damage and cell death.

Tumor suppressor WWOX moderates the mitochondrial respiratory complex.

Fragile site FRA16D exhibits DNA instability in cancer, resulting in diminished levels of protein from the WWOX gene that spans it. WWOX suppresses tumor growth by an undefined mechanism. WWOX participates in pathways involving aerobic metabolism and reactive oxygen species. WWOX comprises two WW domains as well as a short-chain dehydrogenase/reductase enzyme. Herein is described an in vivo genetic analysis in Drosophila melanogaster to identify functional interactions between WWOX and metabolic pathways. Altered WWOX levels modulate variable cellular outgrowths caused by genetic deficiencies of components of the mitochondrial respiratory complexes. This modulation requires the enzyme active site of WWOX, and the defective respiratory complex-induced cellular outgrowths are mediated by reactive oxygen species, dependent upon the Akt pathway and sensitive to levels of autophagy and hypoxia-inducible factor. WWOX is known to contribute to homeostasis by regulating the balance between oxidative phosphorylation and glycolysis. Reduction of WWOX levels results in diminished ability to respond to metabolic perturbation of normal cell growth. Thus, the ability of WWOX to facilitate escape from mitochondrial damage-induced glycolysis (Warburg effect) is, therefore, a plausible mechanism for its tumor suppressor activity.

Tumor Suppressor WWOX Contributes to the Elimination of Tumorigenic Cells in Drosophila melanogaster.

WWOX is a >1 Mb gene spanning FRA16D Common Chromosomal Fragile Site, a region of DNA instability in cancer. Consequently, altered WWOX levels have been observed in a wide variety of cancers. In vitro studies have identified a large number and variety of potential roles for WWOX. Although its normal role in vivo and functional contribution to cancer have not been fully defined, WWOX does have an integral role in metabolism and can suppress tumor growth. Using Drosophila melanogaster as an in vivo model system, we find that WWOX is a modulator of TNFα/Egr-mediated cell death. We found that altered levels of WWOX can modify phenotypes generated by low level ectopic expression of TNFα/Egr and this corresponds to altered levels of Caspase 3 activity. These results demonstrate an in vivo role for WWOX in promoting cell death. This form of cell death is accompanied by an increase in levels of reactive oxygen species, the regulation of which we have previously shown can also be modified by altered WWOX activity. We now hypothesise that, through regulation of reactive oxygen species, WWOX constitutes a link between alterations in cellular metabolism observed in cancer cells and their ability to evade normal cell death pathways. We have further shown that WWOX activity is required for the efficient removal of tumorigenic cells from a developing epithelial tissue. Together these results provide a molecular basis for the tumor suppressor functions of WWOX and the better prognosis observed in cancer patients with higher levels of WWOX activity. Understanding the conserved cellular pathways to which WWOX contributes provides novel possibilities for the development of therapeutic approaches to restore WWOX function in cancer.

WWOX, the chromosomal fragile site FRA16D spanning gene: its role in metabolism and contribution to cancer.

The WWOX gene spans the common chromosomal fragile site FRA16D that is located within a massive (780 kb) intron. The WWOX gene is very long, at 1.1 Mb, which may contribute to the very low abundance of the full-length 1.4 kb mRNA. Alternative splicing also accounts for a variety of aberrant transcripts, most of which are devoid of C-terminal sequences required for WWOX to act as an oxidoreductase. The mouse WWOX gene also spans a chromosomal fragile site implying some sort of functional relationship that confers a selective advantage. The encoded protein domains of WWOX are conserved through evolution (between humans and Drosophila melanogaster) and include WW domains, an NAD -binding site, short-chain dehydrogenase/reductase enzyme and nuclear compartmentalization signals. This homology has enabled functional analyses in D. melanogaster that demonstrate roles for WWOX in reactive oxygen species regulation and metabolism. Indeed the human WWOX gene is also responsive to altered metabolism. Cancer cells typically exhibit altered metabolism (Warburg effect). Many cancers exhibit FRA16D DNA instability that results in aberrant WWOX expression and is associated with poor prognosis for these cancers. It is therefore thought that aberrant WWOX expression contributes to the altered metabolism in cancer. In addition, others have found that a specific (low-expression) allele of WWOX genotype contributes to cancer predisposition.

RNA pathogenesis via Toll-like receptor-activated inflammation in expanded repeat neurodegenerative diseases.

Previously, we hypothesized that an RNA-based pathogenic pathway has a causal role in the dominantly inherited unstable expanded repeat neurodegenerative diseases. In support of this hypothesis we, and others, have characterized rCAG.rCUG 100 repeat double-strand RNA (dsRNA) as a previously unidentified agent capable of causing pathogenesis in a Drosophila model of neurodegenerative disease. Dicer, Toll, and autophagy pathways have distinct roles in this Drosophila dsRNA pathology. Dicer dependence is accompanied by cleavage of rCAG.rCUG 100 repeat dsRNA down to r(CAG) 7 21-mers. Among the "molecular hallmarks" of this pathway that have been identified in Drosophila, some [i.e., r(CAG) 7 and elevated tumor necrosis factor] correlate with observations in affected people (e.g., Huntington's disease and amyotrophic lateral sclerosis) or in related animal models (i.e., autophagy). The Toll pathway is activated in the presence of repeat-containing dsRNA and toxicity is also dependent on this pathway. How might the endogenously expressed dsRNA mediate Toll-dependent toxicity in neuronal cells? Endogenous RNAs are normally shielded from Toll pathway activation as part of the mechanism to distinguish "self" from "non-self" RNAs. This typically involves post-transcriptional modification of the RNA. Therefore, it is likely that rCAG.rCUG 100 repeat dsRNA has a characteristic property that interferes with or evades this normal mechanism of shielding. We predict that repeat expansion leads to an alteration in RNA structure and/or form that perturbs RNA modification, causing the unshielded repeat RNA (in the form of its Dicer-cleaved products) to be recognized by Toll-like receptors (TLRs), with consequent activation of the Toll pathway leading to loss of cell function and then ultimately cell death. We hypothesize that the proximal cause of expanded repeat neurodegenerative diseases is the TLR recognition (and resultant innate inflammatory response) of repeat RNA as "non-self" due to their paucity of "self" modification.

Common chromosomal fragile site FRA16D tumor suppressor WWOX gene expression and metabolic reprograming in cells.

The WWOX gene spans the FRA16D common chromosomal fragile site and is able to suppress tumor growth. FRA16D is a frequent site of DNA instability in cancer resulting in reduced levels of WWOX expression. Altered levels of WWOX have been shown to affect metabolism. Whereas metabolic reprograming of cells from oxidative phosphorylation to aerobic glycolysis is a major hallmark of tumors, the relationship between common chromosomal fragile site genes and altered metabolism has been unclear. Here we report that altering metabolism from glycolysis to oxidative phosphorylation causes stable increase in steady-state levels of transcripts of the WWOX gene. Consistent with this, exposure to hypoxic conditions, in which cells rely on glycolysis, causes a downregulation of WWOX mRNA. The function of WWOX is therefore intimately integrated with metabolism, as WWOX not only contributes to the metabolic state of cells, its transcript levels are also linked to intracellular metabolic state.

Distinct roles for Toll and autophagy pathways in double-stranded RNA toxicity in a Drosophila model of expanded repeat neurodegenerative diseases.

Dominantly inherited expanded repeat neurodegenerative diseases are caused by the expansion of variable copy number tandem repeat sequences in otherwise unrelated genes. Some repeats encode polyglutamine that is thought to be toxic; however, other repeats do not encode polyglutamine indicating either multiple pathogenic pathways or an alternative common toxic agent. As these diseases share numerous clinical features and expanded repeat RNA is a common intermediary, RNA-based pathogenesis has been proposed, based on its toxicity in animal models. In Drosophila, double-stranded (rCAG.rCUG∼100) RNA toxicity is Dicer dependent and generates single-stranded (rCAG)7, an entity also detected in affected Huntington's Disease (HD) brains. We demonstrate that Drosophila rCAG.rCUG∼100 RNA toxicity perturbs several pathways including innate immunity, consistent with the observation in HD that immune activation precedes neuronal toxicity. Our results show that Drosophila rCAG.rCUG∼100 RNA toxicity is dependent upon Toll signaling and sensitive to autophagy, further implicating innate immune activation. In exhibiting molecular and cellular hallmarks of HD, double-stranded RNA-mediated activation of innate immunity is, therefore, a candidate pathway for this group of human genetic diseases.

Ubiquitous expression of CUG or CAG trinucleotide repeat RNA causes common morphological defects in a Drosophila model of RNA-mediated pathology.

Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington's disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their 'molecular hallmarks' that can be used to determine the contribution of each pathway in human pathology. Here we characterise a tergite disruption phenotype in adult flies, caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait we define a new genetic system in which to examine 'hairpin' repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA-mediated pathology in other model systems, contribute to cellular perturbation in this model.

Dynamic mutations: where are they now?

Dynamic mutations are those caused by the expansion of existing polymorphic DNA repeat sequences beyond a copy number threshold. These genetic mutations can give rise to dominant, recessive or X-linked disorders, dependent upon the location of the repeat sequence with respect to the genes that are affected by the expansion. The distinguishing feature of these mutations is their instability, which is a function of the copy number of repeats and can occur in either meiosis or mitosis. For some of the resultant disorders there is a relationship between repeat copy number and age-at-onset and/or severity ofsymptoms ofthe disease. For this reason much effort is now focused on identifying the pathogenic pathways from the mutation to the disease symptoms in the hope of finding means of delaying onset, slowing progression or even preventing symptoms ofthe disease. The growing list ofneurodegenerative and neuromuscular diseases caused by dynamic mutations includes Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), a number of spinocerebellar ataxias (SCAs), oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy Type 1 and 2 (DM1 and 2), Huntington's disease-like 2 (HDL-2), Friedrich's ataxia (FRDA), Fragile X associated tremor ataxia syndrome (FXTAS), Fragile XE (FRAXE) and Fragile XA (FRAXA). This chapter aims to give a brief overview of what is currently known about each disease and the mechanisms underlying pathogenesis.

Comparative toxicity of polyglutamine, polyalanine and polyleucine tracts in Drosophila models of expanded repeat disease.

Homopolymeric amino acid repeat sequences in proteins are of particular interest due to the discovery that expanded copy numbers of these repeats are the molecular basis for a growing list of human genetic diseases. Repeat copy numbers above a typical normal range of polyglutamine repeats have been found to be the principal pathogenic agents in a number of these diseases, including Huntington's disease. There is emerging evidence that expansions of amino acids encoded by other reading frames of CAG/CUG repeats, including polyalanine and polyleucine, could contribute to toxicity in the 'polyglutamine' diseases. We have therefore used the Drosophila model system to investigate effects of ectopic expression of polyglutamine, polyleucine and polyalanine repeats in vivo to assess their relative toxicities and the common and distinct characteristics of the pathogenesis that they cause. We find that these homopolymeric sequences all exhibit toxicity and are able to form aggregates in Drosophila, although there are marked differences in the degree of toxicity dependent upon the tissue in which they are expressed.

Double-stranded RNA is pathogenic in Drosophila models of expanded repeat neurodegenerative diseases.

The pathogenic agent responsible for the expanded repeat diseases, a group of neurodegenerative diseases that includes Huntington's disease is not yet fully understood. Expanded polyglutamine (polyQ) is thought to be the toxic agent in certain cases, however, not all expanded repeat disease genes can encode a polyQ sequence. Since a repeat-containing RNA intermediary is common to all of these diseases, hairpin-forming single-stranded RNA has been investigated as a potential common pathogenic agent. More recently, it has become apparent that most of the expanded repeat disease loci have transcription occurring from both strands, raising the possibility that the complementary repeat RNAs could form a double-stranded structure. In our investigation using Drosophila models of these diseases, we identified a fortuitous integration event that models bidirectional repeat RNA transcription with the resultant flies exhibiting inducible pathology. We therefore established further lines of Drosophila expressing independent complementary repeat RNAs and found that these are toxic. The Dicer pathway is essential for this toxicity and in neuronal cells accounts for metabolism of the high copy number (CAG.CUG)(100) double-stranded RNAs down to (CAG)(7) single-stranded small RNAs. We also observe significant changes to the microRNA profile in neurons. These data identify a novel pathway through which double-stranded repeat RNA is toxic and capable of eliciting symptoms common to neurodegenerative human diseases resulting from dominantly inherited expanded repeats.

Perturbation of the Akt/Gsk3-ß signalling pathway is common to Drosophila expressing expanded untranslated CAG, CUG and AUUCU repeat RNAs.

Recent evidence supports a role for RNA as a common pathogenic agent in both the 'polyglutamine' and 'untranslated' dominant expanded repeat disorders. One feature of all repeat sequences currently associated with disease is their predicted ability to form a hairpin secondary structure at the RNA level. In order to investigate mechanisms by which hairpin-forming repeat RNAs could induce neurodegeneration, we have looked for alterations in gene transcript levels as hallmarks of the cellular response to toxic hairpin repeat RNAs. Three disease-associated repeat sequences--CAG, CUG and AUUCU--were specifically expressed in the neurons of Drosophila and resultant common transcriptional changes assessed by microarray analyses. Transcripts that encode several components of the Akt/Gsk3-ß signalling pathway were altered as a consequence of expression of these repeat RNAs, indicating that this pathway is a component of the neuronal response to these pathogenic RNAs and may represent an important common therapeutic target in this class of diseases.

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species.

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a significant proportion of Drosophila Wwox interactors identified by proteomics and microarray analyses have roles in aerobic metabolism. Functional relationships between Wwox and either CG6439/isocitrate dehydrogenase (Idh) or Cu-Zn superoxide dismutase (Sod) were confirmed by genetic interactions. In addition, altered levels of Wwox resulted in altered levels of endogenous reactive oxygen species. Wwox (like FHIT) contributes to pathways involving aerobic metabolism and oxidative stress, providing an explanation for the 'non-classical tumour suppressor' behaviour of WWOX. Fragile sites, and the genes that span them, are therefore part of a protective response mechanism to oxidative stress and likely contributors to the differences seen in aerobic glycolysis (Warburg effect) in cancer cells.

Selective neuronal requirement for huntingtin in the developing zebrafish.

Huntington's disease shares a common molecular basis with eight other neurodegenerative diseases, expansion of an existing polyglutamine tract. In each case, this repeat tract occurs within otherwise unrelated proteins. These proteins show widespread and overlapping patterns of expression in the brain and yet the diseases are distinguished by neurodegeneration in a specific subset of neurons that are most sensitive to the mutation. It has therefore been proposed that expansion of the polyglutamine region in these genes may result in perturbation of the normal function of the respective proteins, and that this perturbation in some way contributes to the neuronal specificity of these diseases. The normal functions of these proteins have therefore become a focus for investigation as potential pathogenic pathways. We have used synthetic antisense morpholinos to inhibit the translation of huntingtin mRNA during early zebrafish development and have previously reported the effects of huntingtin reduction on iron transport and homeostasis. Here we report an analysis of the effects of huntingtin loss-of-function on the developing nervous system, observing distinct defects in morphology of neuromasts, olfactory placode and branchial arches. The potential common origins of these defects were explored, revealing impaired formation of the anterior-most region of the neural plate as indicated by reduced pre-placodal and telencephalic gene expression with no effect on mid- or hindbrain formation. These investigations demonstrate a specific 'rate-limiting' role for huntingtin in formation of the telencephalon and the pre-placodal region, and differing levels of requirement for huntingtin function in specific nerve cell types.