Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when the effects of homozygosity for recessive mutations are studied in non-isogenic backgrounds, genes located proximal to the mutation on the same chromosome often appear over-represented among those genes identified as differentially expressed (DE). One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect functional responses to the mutation but, instead, result from an unequal distribution of expression quantitative trait loci (eQTLs) between sample groups of mutant or wild-type genotypes. This is problematic because eQTL expression differences are difficult to distinguish from genes that are DE due to functional responses to a mutation. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) are also observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between those sample groups subjected to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting-based approaches. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. Our method for DAR analysis requires only RNA-sequencing data, facilitating its application across new and existing datasets.

Accelerated loss of hypoxia response in zebrafish with familial Alzheimer's disease-like mutation of presenilin 1.

Ageing is the major risk factor for Alzheimer's disease (AD), a condition involving brain hypoxia. The majority of early-onset familial AD (EOfAD) cases involve dominant mutations in the gene PSEN1. PSEN1 null mutations do not cause EOfAD. We exploited putative hypomorphic and EOfAD-like mutations in the zebrafish psen1 gene to explore the effects of age and genotype on brain responses to acute hypoxia. Both mutations accelerate age-dependent changes in hypoxia-sensitive gene expression supporting that ageing is necessary, but insufficient, for AD occurrence. Curiously, the responses to acute hypoxia become inverted in extremely aged fish. This is associated with an apparent inability to upregulate glycolysis. Wild-type PSEN1 allele expression is reduced in post-mortem brains of human EOfAD mutation carriers (and extremely aged fish), possibly contributing to EOfAD pathogenesis. We also observed that age-dependent loss of HIF1 stabilization under hypoxia is a phenomenon conserved across vertebrate classes.

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer's disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis.

BACKGROUND: Early-onset familial Alzheimer's disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer's disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. RESULTS: We generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. CONCLUSIONS: Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3' untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

Alzheimer's disease-related peptide PS2V plays ancient, conserved roles in suppression of the unfolded protein response under hypoxia and stimulation of γ-secretase activity.

The PRESENILIN1 and PRESENILIN2 genes encode structurally related proteases essential for γ-secretase activity. Of nearly 200 PRESENILIN mutations causing early onset, familial Alzheimer's disease (FAD) only the K115Efx10 mutation of PSEN2 causes truncation of the open reading frame. If translated, the truncated product would resemble a naturally occurring isoform of PSEN2 named PS2V that is induced by hypoxia and found at elevated levels in late onset Alzheimer's disease (AD) brains. The function of PS2V is largely unexplored. We show that zebrafish possess a PS2V-like isoform, PS1IV, produced from the fish's PSEN1 rather than PSEN2 orthologous gene. The molecular mechanism controlling formation of PS2V/PS1IV was probably present in the ancient common ancestor of the PSEN1 and PSEN2 genes. Human PS2V and zebrafish PS1IV have highly divergent structures but conserved abilities to stimulate γ-secretase activity and to suppress the unfolded protein response (UPR) under hypoxia. The putative protein truncation caused by K115Efx10 resembles PS2V in its ability to increase γ-secretase activity and suppress the UPR. This supports increased Aß levels as a common link between K115Efx10 early onset AD and sporadic, late onset AD. The ability of mutant variants of PS2V to stimulate γ-secretase activity partially correlates with their ability to suppress the UPR. The cytosolic, transmembrane and luminal domains of PS2V are all critical to its γ-secretase and UPR-suppression activities. Our data support a model in which chronic hypoxia in aged brains promotes excessive Notch signalling and accumulation of Aß that contribute to AD pathogenesis.

Differential, dominant activation and inhibition of Notch signalling and APP cleavage by truncations of PSEN1 in human disease.

PRESENILIN1 (PSEN1) is the major locus for mutations causing familial Alzheimer's disease (FAD) and is also mutated in Pick disease of brain, familial acne inversa and dilated cardiomyopathy. It is a critical facilitator of Notch signalling and many other signalling pathways and protein cleavage events including production of the Amyloidß (Aß) peptide from the AMYLOID BETA A4 PRECURSOR PROTEIN (APP). We previously reported that interference with splicing of transcripts of the zebrafish orthologue of PSEN1 creates dominant negative effects on Notch signalling. Here, we extend this work to show that various truncations of human PSEN1 (or zebrafish Psen1) protein have starkly differential effects on Notch signalling and cleavage of zebrafish Appa (a paralogue of human APP). Different truncations can suppress or stimulate Notch signalling but not Appa cleavage and vice versa. The G183V mutation possibly causing Pick disease causes production of aberrant transcripts truncating the open reading frame after exon 5 sequence. We show that the truncated protein potentially translated from these transcripts avidly incorporates into very stable Psen1-dependent higher molecular weight complexes and suppresses cleavage of Appa but not Notch signalling. In contrast, the truncated protein potentially produced by the P242LfsX11 acne inversa mutation has no effect on Appa cleavage but, unexpectedly, enhances Notch signalling. Our results suggest novel hypotheses for the pathological mechanisms underlying these diseases and illustrate the importance of investigating the function of dominant mutations at physiologically relevant expression levels and in the normally heterozygous state in which they cause human disease rather than in isolation from healthy alleles.

Analysis of nicastrin gene phylogeny and expression in zebrafish.

NICASTRIN is a component of the aspartyl protease γ-secretase complex which is involved in intramembranous cleavage of type I transmembrane proteins, notably the Notch receptor proteins and the AMYLOID BETA A4 PRECURSOR PROTEIN (APP). This study aimed to characterize the orthologue of the human NICASTRIN (NCSTN) gene in zebrafish, an advantageous model organism for the study of human disease. Zebrafish Nicastrin protein was predicted to possess the conserved glutamate 333 residue and DYIGS motif of human NCSTN that are important for substrate recognition/processing in γ-secretase. Quantitative real-time RT-PCR revealed the profile of relative zebrafish nicastrin (ncstn) transcript levels in embryos at different times during development and in adult tissues. The analysis of synteny conservation revealed local rearrangements of ncstn and another gene, mpz, relative to copa, and pex19. In situ hybridization showed higher relative levels of ncstn transcripts in the developing brain and otic vesicles of embryos at 24 and 48 h post fertilization, respectively. Our observations are consistent with a role for Ncstn protein in Notch signaling within the proliferative ventricular zone of the developing central nervous system.

Identification and expression analysis of the zebrafish orthologues of the mammalian MAP1LC3 gene family.

Autophagy is the principle pathway within cells involved in clearing damaged proteins and organelles. Therefore autophagy is necessary to maintain the turnover balance of peptides and homoeostasis. Autophagy occurs at basal levels under normal conditions but can be upregulated by chemical inducers or stress conditions. The zebrafish (Danio rerio) serves as a versatile tool to understand the functions of genes implicated in autophagy. We report the identification of the zebrafish orthologues of mammalian genes MAP1LC3A (map1lc3a) and MAP1LC3B (map1lc3b) by phylogenetic and conserved synteny analysis and we examine their expression during embryonic development. The zebrafish map1lc3a and map1lc3b genes both show maternally contributed transcripts in early embryogenesis. However, levels of map1lc3a transcript steadily increase until at least 120h post-fertilisation while the levels of map1lc3b show a more variable pattern across developmental time. We have also validated the LC3I ratio/LC3I immunoblot autophagy assay in the presence of chloroquine (a lysosomal proteolysis inhibitor). We found that the LC3II/LC3I ratio is significantly increased in the presence of sodium azide with chloroquine supporting that hypoxia induces autophagy in zebrafish. This was supported by our qPCR assay that showed increased map1lc3a transcript levels in the presence of sodium azide. In contrast, levels of map1lc3b transcripts were reduced in the presence of rapamycin but the decrease in the presence of sodium azide did not reach statistical significance. Our study supports the use of zebrafish for analysing the interplay between hypoxia, development and autophagy.

An Alternative View of Familial Alzheimer's Disease Genetics.

Probabilistic and parsimony-based arguments regarding available genetics data are used to propose that Hardy and Higgin's amyloid cascade hypothesis is valid but is commonly interpreted too narrowly to support, incorrectly, the primacy of the amyloid-ß peptide (Aß) in driving Alzheimer's disease pathogenesis. Instead, increased activity of the ßCTF (C99) fragment of AßPP is the critical pathogenic determinant altered by mutations in the APP gene. This model is consistent with the regulation of APP mRNA translation via its 5' iron responsive element. Similar arguments support that the pathological effects of familial Alzheimer's disease mutations in the genes PSEN1 and PSEN2 are not exerted directly via changes in AßPP cleavage to produce different ratios of Aß length. Rather, these mutations likely act through effects on presenilin holoprotein conformation and function, and possibly the formation and stability of multimers of presenilin holoprotein and/or of the γ-secretase complex. All fAD mutations in APP, PSEN1, and PSEN2 likely find unity of pathological mechanism in their actions on endolysosomal acidification and mitochondrial function, with detrimental effects on iron homeostasis and promotion of "pseudo-hypoxia" being of central importance. Aß production is enhanced and distorted by oxidative stress and accumulates due to decreased lysosomal function. It may act as a disease-associated molecular pattern enhancing oxidative stress-driven neuroinflammation during the cognitive phase of the disease.

pHAPE: a plasmid for production of DNA size marker ladders for gel electrophoresis.

DNA size markers (also known as 'molecular weight markers' or 'DNA ladders') are an essential tool when using gel electrophoresis to identify and purify nucleic acids. However, the cost of these DNA ladders is not insignificant and, over time, impinges on the funds available for research and training in molecular biology. Here, we describe a method for the generation of 'pHAPE', a plasmid from which a variety of DNA ladders can be generated via simple restriction enzyme digestions. The pHAPE plasmid can be generated by mutagenesis of the commonly used pBluescript II SK+ phagemid followed by insertion of a 7141 bp sequence (comprised of three smaller, synthetic fragments). Our use of pHAPE allows us some small relief from the ever-rising costs of performing molecular biology experiments ('Don't worry, pHAPE').

Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when homozygous mutations are studied in non-isogenic backgrounds, genes from the same chromosome as a mutation often appear over-represented among differentially expressed (DE) genes. One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect true biological responses to the mutation but, instead, result from differences in representation of expression quantitative trait loci (eQTLs) between sample groups selected on the basis of mutant or wild-type genotype. This is problematic when inclusion of spurious DE genes in a functional enrichment study results in incorrect inferences of mutation effect. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) can also be observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between groups of samples subject to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting of gene-level rankings. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. The DAR metric provides a solid foundation for addressing the eQTL issue in new and existing datasets because it relies solely on RNA-sequencing data.

The Use of Zebrafish in Transcriptome Analysis of the Early Effects of Mutations Causing Early Onset Familial Alzheimer's Disease and Other Inherited Neurodegenerative Conditions.

The degree to which non-human animals can be used to model Alzheimer's disease is a contentious issue, particularly as there is still widespread disagreement regarding the pathogenesis of this neurodegenerative dementia. The currently popular transgenic models are based on artificial expression of genes mutated in early onset forms of familial Alzheimer's disease (EOfAD). Uncertainty regarding the veracity of these models led us to focus on heterozygous, single mutations of endogenous genes (knock-in models) as these most closely resemble the genetic state of humans with EOfAD, and so incorporate the fewest assumptions regarding pathological mechanism. We have generated a number of lines of zebrafish bearing EOfAD-like and non-EOfAD-like mutations in genes equivalent to human PSEN1, PSEN2, and SORL1. To analyze the young adult brain transcriptomes of these mutants, we exploited the ability of zebrafish to produce very large families of simultaneous siblings composed of a variety of genotypes and raised in a uniform environment. This "intra-family" analysis strategy greatly reduced genetic and environmental "noise" thereby allowing detection of subtle changes in gene sets after bulk RNA sequencing of entire brains. Changes to oxidative phosphorylation were predicted for all EOfAD-like mutations in the three genes studied. Here we describe some of the analytical lessons learned in our program combining zebrafish genome editing with transcriptomics to understand the molecular pathologies of neurodegenerative disease.

Aicardi Syndrome Is a Genetically Heterogeneous Disorder.

Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile epileptic spasms syndrome. The diagnostic criteria of AIC were revised in 2005 to include additional phenotypes that are frequently observed in this patient group. AIC has been traditionally considered as X-linked and male lethal because it almost exclusively affects females. Despite numerous genetic and genomic investigations on AIC, a unifying X-linked cause has not been identified. Here, we performed exome and genome sequencing of 10 females with AIC or suspected AIC based on current criteria. We identified a unique de novo variant, each in different genes: KMT2B, SLF1, SMARCB1, SZT2 and WNT8B, in five of these females. Notably, genomic analyses of coding and non-coding single nucleotide variants, short tandem repeats and structural variation highlighted a distinct lack of X-linked candidate genes. We assessed the likely pathogenicity of our candidate autosomal variants using the TOPflash assay for WNT8B and morpholino knockdown in zebrafish (Danio rerio) embryos for other candidates. We show expression of Wnt8b and Slf1 are restricted to clinically relevant cortical tissues during mouse development. Our findings suggest that AIC is genetically heterogeneous with implicated genes converging on molecular pathways central to cortical development.

Zebrafish as a tool in Alzheimer's disease research.

Alzheimer's disease is the most prevalent form of neurodegenerative disease. Despite many years of intensive research our understanding of the molecular events leading to this pathology is far from complete. No effective treatments have been defined and questions surround the validity and utility of existing animal models. The zebrafish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural structure and genome. Zebrafish genes orthologous to those mutated in human familial Alzheimer's disease have been defined. Work in zebrafish has permitted discovery of unique characteristics of these genes that would have been difficult to observe with other models. In this brief review we give an overview of Alzheimer's disease and transgenic animal models before examining the current contribution of zebrafish to this research area. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.

The protein tyrosine phosphatase Pez regulates TGFbeta, epithelial-mesenchymal transition, and organ development.

Epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.

The response of HMGA1 to changes in oxygen availability is evolutionarily conserved.

Zebrafish embryos have evolved to cope with hypoxia during development. This includes the ability to completely suspend embryo development for extended periods until normoxia is restored. However, only a limited number of studies have examined the gene regulatory responses of zebrafish embryos to hypoxia. The High Mobility Group A1 protein encoded by the mammalian gene HMGA1 is widely expressed during embryo development but not in adults. Its expression can be induced in adult neurons by hypoxia/oxidative stress and it is commonly reactivated in many types of cancer. We report the identification by phylogenetic and conserved synteny analyses of an HMGA1 orthologue in zebrafish, hmga1 (hmg-i/y) and analysis of sodium azide as a chemical agent for inducing hypoxia-like responses in zebrafish embryos including temporary suspension of development ("suspended animation"). Evidence was only found for the existence of the "a" isoform of HMGA1 in fish. The "b" and "c" isoforms were not detected. We show that zebrafish hmga1 is expressed in a manner similar to in mammals including its induction by hypoxia during hatching stage and in adult zebrafish brain. However, earlier during development, hypoxia causes a decrease in hmga1 transcript levels. By analysis of conservation of the HMGA1a isoform binding site in zebrafish psen2 gene transcripts, we predict that a zebrafish equivalent of the PS2V isoform of human PSEN2 is not formed and we support this by RT-PCR analyses. Thus, analysis of hmga1 function in zebrafish embryogenesis may be valuable for understanding its wider role in vertebrate development, cancer and cellular responses to hypoxia but not for analysis of the action of HMGA1 in PS2V formation.

Brain transcriptomes of zebrafish and mouse Alzheimer's disease knock-in models imply early disrupted energy metabolism.

Energy production is the most fundamentally important cellular activity supporting all other functions, particularly in highly active organs, such as brains. Here, we summarise transcriptome analyses of young adult (pre-disease) brains from a collection of 11 early-onset familial Alzheimer's disease (EOFAD)-like and non-EOFAD-like mutations in three zebrafish genes. The one cellular activity consistently predicted as affected by only the EOFAD-like mutations is oxidative phosphorylation, which produces most of the energy of the brain. All the mutations were predicted to affect protein synthesis. We extended our analysis to knock-in mouse models of APOE alleles and found the same effect for the late onset Alzheimer's disease risk allele ε4. Our results support a common molecular basis for the initiation of the pathological processes leading to both early and late onset forms of Alzheimer's disease, and illustrate the utility of zebrafish and knock-in single EOFAD mutation models for understanding the causes of this disease.

Identification and expression analysis of the zebrafish orthologue of Klotho.

Klotho is an aging suppressor gene. In mice, loss of Klotho function causes accelerated aging while increased Klotho expression increases longevity. This study aimed to identify and characterize the orthologue of Klotho in zebrafish, a powerful model organism for the investigation of development and human disease. Zebrafish klotho was identified by a bioinformatics approach, and cloning and sequencing of klotho cDNA confirmed the in silico analysis. The zebrafish Klotho protein has a structure similar to human and mouse Klotho, but it lacks an apparent secretory signal sequence. We can find no evidence of an alternative transcript isoform lacking the transmembrane domain coding sequence as seen in mammals. RT-PCR revealed the expression of klotho during embryonic development and in a wider variety of adult tissues than in mouse. Quantitative real-time RT-PCR demonstrated the relative gene expression profile of zebrafish Klotho during embryogenesis and in adult tissues. In situ hybridization showed an apparently diffuse signal of klotho mRNA expression in the adult zebrafish testis.

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.

Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord.

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine "irregular" patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.

Iron Responsive Element-Mediated Responses to Iron Dyshomeostasis in Alzheimer's Disease.

BACKGROUND: Iron trafficking and accumulation is associated with Alzheimer's disease (AD) pathogenesis. However, the role of iron dyshomeostasis in early disease stages is uncertain. Currently, gene expression changes indicative of iron dyshomeostasis are not well characterized, making it difficult to explore these in existing datasets. OBJECTIVE: To identify sets of genes predicted to contain iron responsive elements (IREs) and use these to explore possible iron dyshomeostasis-associated gene expression responses in AD. METHODS: Comprehensive sets of genes containing predicted IRE or IRE-like motifs in their 3' or 5' untranslated regions (UTRs) were identified in human, mouse, and zebrafish reference transcriptomes. Further analyses focusing on these genes were applied to a range of cultured cell, human, mouse, and zebrafish gene expression datasets. RESULTS: IRE gene sets are sufficiently sensitive to distinguish not only between iron overload and deficiency in cultured cells, but also between AD and other pathological brain conditions. Notably, changes in IRE transcript abundance are among the earliest observable changes in zebrafish familial AD (fAD)-like brains, preceding other AD-typical pathologies such as inflammatory changes. Unexpectedly, while some IREs in the 3' untranslated regions of transcripts show significantly increased stability under iron deficiency in line with current assumptions, many such transcripts instead display decreased stability, indicating that this is not a generalizable paradigm. CONCLUSION: Our results reveal IRE gene expression changes as early markers of the pathogenic process in fAD and are consistent with iron dyshomeostasis as an important driver of this disease. Our work demonstrates how differences in the stability of IRE-containing transcripts can be used to explore and compare iron dyshomeostasis-associated gene expression responses across different species, tissues, and conditions.