Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

Temple University 

External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors.

Efficient disruption of Zebrafish genes using a Gal4-containing gene trap.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia in the industrialised world. The two monoamine oxidase (MAO) enzymes, monoamine oxidase A (MAOA) and monoamine oxidase B (MAOB), are important in the metabolism of monoamine neurotransmitters. AD and ageing have been shown to increase enzyme activity for both MAOA and MAOB. An increase (rather than decrease) of enzyme activity is a rare event in a disease that results in a decrease in the number of cells in the brain. The mechanism, transcriptional or post-transcriptional, responsible for the increase in protein activity, is not known. In this study, we investigate for the first time the messenger RNA (mRNA) expression levels of both MAOA and MAOB in 246 cortical brain samples obtained at autopsy from 62 AD patients and 61 normal controls. We found a significant increase in mRNA levels for both MAOA (P=0.001) and MAOB (P=0.002) in disease brain tissue. This indicates that both MAO enzymes might be important in the progression of AD.

Increased monoamine oxidase messenger RNA expression levels in frontal cortex of Alzheimer's disease patients.

Proteins containing the DM domain, a zinc finger-like DNA binding motif, have been implicated in sexual differentiation in diverse metazoan organisms. Of seven mammalian DM domain genes, only Dmrt1 and Dmrt2 have been functionally analyzed. Here, we report expression analysis and targeted disruption of Dmrt4 (also called DmrtA1) in the mouse. Dmrt4 is widely expressed during embryonic and postnatal development. However, we find that mice homozygous for a putative null mutation in Dmrt4 develop essentially normally, undergo full sexual differentiation in both sexes, and are fertile. We observed two potential mutant phenotypes in Dmrt4 mutant mice. First, ovaries of most mutant females have polyovular follicles, suggesting a role in folliculogenesis. Second, 25% of mutant males consistently exhibited copulatory behavior toward other males. We also tested potential redundancy between Dmrt4 and two other gonadally expressed DM domain genes, Dmrt1 and Dmrt7. We observed no enhancement of gonadal phenotypes in the double mutants, suggesting that these genes function independently in gonadal development.

Mice mutant in the DM domain gene Dmrt4 are viable and fertile but have polyovular follicles.

Low-copy repeats (LCRs) are genomic features that affect chromosome stability and can produce disease-associated rearrangements. We describe members of three families with deletions in 10q22.3-q23.31, a region harboring a complex set of LCRs, and demonstrate that rearrangements in this region are associated with behavioral and neurodevelopmental abnormalities, including cognitive impairment, autism, hyperactivity, and possibly psychiatric disease. Fine mapping of the deletions in members of all three families by use of a custom 10q oligonucleotide array-based comparative genomic hybridization (NimbleGen) and polymerase chain reaction-based methods demonstrated a different deletion in each family. In one proband, the deletion breakpoints are associated with DNA fragments containing noncontiguous sequences of chromosome 10, whereas, in the other two families, the breakpoints are within paralogous LCRs, removing approximately 7.2 Mb and 32 genes. Our data provide evidence that the 10q22-q23 genomic region harbors one or more genes important for cognitive and behavioral development and that recurrent deletions affecting this interval define a novel genomic disorder.

Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities.

The genomic architecture of the 10q22q23 region is characterised by two low-copy repeats (LCRs3 and 4), and deletions in this region appear to be rare. We report the clinical and molecular characterisation of eight novel deletions and six duplications within the 10q22.3q23.3 region. Five deletions and three duplications occur between LCRs3 and 4, whereas three deletions and three duplications have unique breakpoints. Most of the individuals with the LCR3-4 deletion had developmental delay, mainly affecting speech. In addition, macrocephaly, mild facial dysmorphisms, cerebellar anomalies, cardiac defects and congenital breast aplasia were observed. For congenital breast aplasia, the NRG3 gene, known to be involved in early mammary gland development in mice, is a putative candidate gene. For cardiac defects, BMPR1A and GRID1 are putative candidate genes because of their association with cardiac structure and function. Duplications between LCRs3 and 4 are associated with variable phenotypic penetrance. Probands had speech and/or motor delays and dysmorphisms including a broad forehead, deep-set eyes, upslanting palpebral fissures, a smooth philtrum and a thin upper lip. In conclusion, duplications between LCRs3 and 4 on 10q22.3q23.2 may lead to a distinct facial appearance and delays in speech and motor development. However, the phenotypic spectrum is broad, and duplications have also been found in healthy family members of a proband. Reciprocal deletions lead to speech and language delay, mild facial dysmorphisms and, in some individuals, to cerebellar, breast developmental and cardiac defects.

The phenotype of recurrent 10q22q23 deletions and duplications.

Children with autism have an elevated frequency of large, rare copy number variants (CNVs). However, the global load of deletions or duplications, per se, and their size, location and relationship to clinical manifestations of autism have not been documented. We examined CNV data from 516 individuals with autism or typical development from the population-based Childhood Autism Risks from Genetics and Environment (CHARGE) study. We interrogated 120 regions flanked by segmental duplications (genomic hotspots) for events >50 kbp and the entire genomic backbone for variants >300 kbp using a custom targeted DNA microarray. This analysis was complemented by a separate study of five highly dynamic hotspots associated with autism or developmental delay syndromes, using a finely tiled array platform (>1 kbp) in 142 children matched for gender and ethnicity. In both studies, a significant increase in the number of base pairs of duplication, but not deletion, was associated with autism. Significantly elevated levels of CNV load remained after the removal of rare and likely pathogenic events. Further, the entire CNV load detected with the finely tiled array was contributed by common variants. The impact of this variation was assessed by examining the correlation of clinical outcomes with CNV load. The level of personal and social skills, measured by Vineland Adaptive Behavior Scales, negatively correlated (Spearman's r = -0.13, P = 0.034) with the duplication CNV load for the affected children; the strongest association was found for communication (P = 0.048) and socialization (P = 0.022) scores. We propose that CNV load, predominantly increased genomic base pairs of duplication, predisposes to autism.

Global increases in both common and rare copy number load associated with autism.

Genetic lineage tracing and conditional mutagenesis are developmental genetics techniques reliant on precise tissue-specific expression of transgenes. In the mouse, high specificity is usually achieved by inserting the transgene into the locus of interest through homologous recombination in embryonic stem cells. In the zebrafish, DNA containing the transgenic construct is randomly integrated into the genome, usually through transposon-mediated transgenesis. Expression of such transgenes is affected by regulatory features surrounding the integration site from general accessibility of chromatin to tissue-specific enhancers. We tested if the 1.2Â kb cHS4 insulators derived from the chicken Î²-globin locus can shield a transgene from chromosomal position effects in the zebrafish genome. As our test promoters, we used two different-length versions of the zebrafish nkx2.5. We found that flanking a transgenic construct by cHS4 insulation sequences leads to overall increase in the expression of nkx2.5:mRFP. However, we also observed a very high degree of variability of mRFP expression, indicating that cHS4 insulators fail to protect nkx2.5:mRFP from falling under the control of enhancers in the vicinity of integration site.

Article	Year
Efficient disruption of Zebrafish genes using a Gal4-containing gene trap. BMC genomics\| PubMed ID: 24034702	2013
Increased monoamine oxidase messenger RNA expression levels in frontal cortex of Alzheimer's disease patients. Neuroscience letters\| PubMed ID: 12052537	2002
Mice mutant in the DM domain gene Dmrt4 are viable and fertile but have polyovular follicles. Molecular and cellular biology\| PubMed ID: 16982677	2006
Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities. American journal of human genetics\| PubMed ID: 17436248	2007
The phenotype of recurrent 10q22q23 deletions and duplications. European journal of human genetics : EJHG\| PubMed ID: 21248748	2011
Global increases in both common and rare copy number load associated with autism. Human molecular genetics\| PubMed ID: 23535821	2013
Chicken Î²-globin insulators fail to shield the nkx2.5 promoter from integration site effects in zebrafish. Molecular genetics and genomics : MGG\| PubMed ID: 24036575	2013

Jorune Balciuniene

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