The ability to easily analyze apoptosis is important in studies of molecular cell biology and to evaluate the relative toxicity of different treatments or environments. This is particularly the case ...when substances such as morpholino oligonucleotides are injected into embryos, as such treatments can cause widespread, complex patterns of apoptosis. Zebrafish embryos are well suited for cell biological and environmental toxicity analyses, but the need remains for a simple method that can analyze levels of apoptosis in a statistically significant number of embryos. Here we present a "group fluorescence" method for rapid, large-scale analysis of relative levels of apoptosis based on densitometric techniques.
The deposition of micro- and nanolitre volumes is crucial in sessile droplet microfluidic systems. Several techniques exist for the fabrication of surfaces with patterned wettabilities; however, many ...of these fabrication techniques are time-consuming and complex. Here, we present a device that allows for deposition of multiple droplets within seconds followed by directed evaporative preconcentration. Hydrophobic-coated glass substrates are fashioned with hydrophilic surface energy traps (SETs) using picosecond laser micromachining. SETs can capture nanolitre volumed droplets of both aqueous and organic liquids through discontinuous dewetting. Modification of the machined hydrophilic shape yields a passive mechanism that preconcentrates analyte through evaporation. Studies and optimizations of SET parameters/dimensions (laser power, laser passes, ring/patch diameter) and their effect on patch wettability and degree of preconcentration are presented. As a demonstration, the optimized platform was used to improve the colourimetric detection of cadmium-containing aqueous samples. The optimized SET design demonstrated an 18-fold increase in colourimetric sensitivity compared to conventional milled SETs, suggesting the design would be well-suited for trace analysis. The evaporative preconcentration was also applied to MALDI-IMS analysis of peptides where it resulted in improved uniformity of deposited analyte and decreased analysis times. The rapid droplet deposition and directed evaporative approach can be tailored to provide different concentration factors and is compatible with a wide variety of detection schemes.
We present a microfluidic platform that rapidly deposits many samples and preconcentrates them, making it suitable for a wide range of high-throughput detection schemes.
Zebrafish aplnra functions in epiboly Nornes, Svanhild; Tucker, Ben; Lardelli, Michael
BMC research notes,
11/2009, Letnik:
2, Številka:
1
Journal Article
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The zebrafish, Danio rerio, possesses the paralogous genes aplnra and aplnrb that are duplicates of an ancestral orthologue of the human APLNR gene encoding a G-protein coupled receptor that binds ...the peptide ligand APELIN and is required for normal cardiovascular function. aplnrb is required for migration of cells contributing to heart development in zebrafish embryos. aplnra is transcribed in a complex pattern during early development but its function in embryogenesis is largely unknown.
Blockage of translation of aplnra mRNA in zebrafish embryos results in retarded or failed epiboly with the blastoderm apparently disconnected from the nuclei of the yolk syncytial layer. Gastrulation is also defective. Failure of correct tail extension is observed with ectopic structures resembling somites positioned dorsal to the spinal cord.
aplnra, unlike its duplicate aplnrb, is essential for normal epiboly, although this function appears to be independent of signalling activated by zebrafish Apelin. The defects in epiboly caused by loss of aplnra activity appear, at least partially, to be due to a requirement for aplnra activity in the yolk syncytial layer.
Fragile X Syndrome is a leading heritable cause of mental retardation that results from the loss of FMR1 gene function. Studies in mouse and Drosophila model organisms have been critical in ...understanding many aspects of the loss of function of the FMR1 gene in the human syndrome. Here, we establish that the zebrafish is a useful model organism for the study of the human fragile X syndrome and can be used to examine phenotypes that are difficult or inaccessible to observation in other model organisms. Using morpholino knockdown of the fmr1 gene, we observed abnormal axonal branching of Rohon–Beard and trigeminal ganglion neurons and guidance and defasciculation defects in the lateral longitudinal fasciculus. We demonstrate that this axonal branching defect can be rescued by treatment with MPEP 2-methyl-6-(phenylethynyl) pyridine. This is consistent with an interaction between mGluR signalling and fmr1 function in neurite morphogenesis. We also describe novel findings of abnormalities in the abundance of trigeminal ganglion neurons and of craniofacial abnormalities apparently due to dysmorphic cartilage formation. These abnormalities may be related to a role for fmr1 in neural crest cell specification and possibly in migration.
Suicide is the second leading cause of death among American Indian and Alaska Native youth, and within the Alaska Native youth subpopulation, the leading cause of death. In response to this public ...health crisis, American Indian and Alaska Native communities have created strategies to protect their young people by building resilience using localized Indigenous well-being frameworks and cultural strengths. These approaches to suicide prevention emphasize promotion of protective factors over risk reduction. A measure of culturally based protective factors from suicide risk has potential to assess outcomes from these strengths-based, culturally grounded suicide prevention efforts, and can potentially address several substantive concerns regarding direct assessment of suicide risk. We report on the Reasons for Life (RFL) scale, a measure of protective factors from suicide, testing psychometric properties including internal structure with 302 rural Alaska Native Yup’ik youth. Confirmatory factor analyses revealed the RFL is best described through three distinct first-order factors organized under one higher second-order factor. Item response theory analyses identified 11 satisfactorily functioning items. The RFL correlates with other measures of more general protective factors. Implications of these findings are described, including generalizability to other American Indian and Alaska Native, other Indigenous, and other culturally distinct suicide disparities groups.
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.
Missense mutations in the PRESENILIN1 (PSEN1) gene frequently underlie familial Alzheimer’s disease (FAD). Nonsense and most splicing mutations result in the synthesis of truncated peptides, and it ...has been assumed that truncated PSEN1 protein is functionless so that heterozygotes for these mutations are unaffected. Some FAD mutations affecting PSEN1 mRNA splicing cause loss of exon 8 or 9 sequences while maintaining the reading frame. We attempted to model these exon-loss mutations in zebrafish embryos by injecting morpholino antisense oligonucleotides (morpholinos) directed against splice acceptor sites in zebrafish psen1 transcripts. However, this produced cryptic changes in splicing potentially forming mRNAs encoding truncated presenilin proteins. Aberrant splicing in the region between exons 6 and 8 produces potent dominant negative effects on Psen1 protein activity, including Notch signalling, and causes a hydrocephalus phenotype. Reductions in Psen1 activity feedback positively to increase psen1 transcription through a mechanism apparently independent of γ-secretase. We present evidence that the dominant negative effects are mediated through production of truncated Psen1 peptides that interfere with the normal activity of both Psen1 and Psen2. Mutations causing such truncations would be dominant lethal in embryo development. Somatic cellular changes in ageing cells that interfere with PSEN1 splicing, or otherwise cause protein truncation, might contribute to sporadic Alzheimer’s disease, cancer and other diseases.
Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of Alzheimer's disease. We have previously described that low-level aberrant ...splicing of exon 8 in zebrafish psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increase psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 of psen2. To determine the molecular etiology of these effects, we performed microarray analyses of global gene expression changes. Of the 100 genes that showed greatest dysregulation after either psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression: cyclin G1 (ccng1), prosaposin (psap), cathepsin Lb (ctslb), heat shock protein 70kDa (hsp70) and hatching enzyme 1 (he1). We used phylogenetic and conserved synteny analysis to confirm the orthology of zebrafish ccng1 with human CCNG1. We analyzed the expression of zebrafish ccng1 in developing embryos to 24 hours post fertilization (hpf). Decreased ccng1 expression does not rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The human fragile X mental retardation syndrome is caused by expansions of a CGG repeat in the FMR1 gene. FXR1 and FXR2 are autosomal paralogs of FMR1. The products of the three genes, FMRP, FXR1P, ...and FXR2P, respectively, belong to a family of RNA-binding proteins. While the FMR1-related gene family is well described in human, mouse and Drosophila, little is known about zebrafish (Danio rerio) orthologs of these genes. Here we collate the known FMR1-related gene sequences from zebrafish, examine their regions of structural conservation, and define their orthologies with the human genes. We demonstrate that zebrafish possess only three FMR1-related genes, fmr1, fxr1 and fxr2, and these are orthologous to the human FMR1, FXR1 and FXR2 genes respectively. We examine the spatiotemporal pattern of transcription of the zebrafish genes from 0 hours post fertilisation (hpf) until 24 hpf. Expression of fmr1, fxr1 and fxr2 is widespread throughout this time. However, relative to surrounding tissues, expression of fxr2 is raised in adaxial and somitic cells by 12 hpf while fxr1 expression is high in the anterior of the embryo, and is raised in adaxial cells by 12 hpf. Distinct patterns (and levels) of expression are seen for the different genes later in development. At 24 hpf, fxr1 and fxr2 transcripts show complex distribution patterns in somites. The expression of the FMR1-related gene family in zebrafish tissues is broadly consistent with expression in mouse and human, supporting the idea that zebrafish should be an excellent model organism in which to study the functions of the vertebrate FMR1-related gene family.
Mouse Usp9x/Fam (fat facets in mouse) and its Drosophila ortholog faf (fat facets) encode substrate-specific deubiquitylating enzymes and are essential for early embryonic development. The zebrafish ...(Danio rerio) is a powerful tool for studying embryonic gene expression patterns and function, and to that end, we sought to characterize the zebrafish Usp9 ortholog. Zebrafish usp9 was identified from database searches, and the predicted Usp9 protein is very highly conserved in mouse (90% identical and 94% similar) over its entire length. Phylogenetic analysis indicated that vertebrate Usp9s are highly clustered and separate from the USP9Y and Drosophila forms. We examined the developmental expression of usp9 from fertilization to 2 days postfertilization. usp9 is initially expressed ubiquitously but later restricted to the cephalic central nervous system, the developing lens, distal tips of the pectoral fin bud, and migrating endoderm. The extraordinary level of conservation between the mouse and zebrafish genes, coupled with equivalent expression patterns, makes zebrafish an appropriate complementary system for the study of usp9 in development.
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