354 research outputs found
Development of a chordate anterior–posterior axis without classical retinoic acid signaling
AbstractDevelopmental signaling by retinoic acid (RA) is thought to be an innovation essential for the origin of the chordate body plan. The larvacean urochordate Oikopleura dioica maintains a chordate body plan throughout life, and yet its genome appears to lack genes for RA synthesis, degradation, and reception. This suggests the hypothesis that the RA-machinery was lost during larvacean evolution, and predicts that Oikopleura development has become independent of RA-signaling. This prediction raises the problem that the anterior–posterior organization of a chordate body plan can be developed without the classical morphogenetic role of RA. To address this problem, we performed pharmacological treatments and analyses of developmental molecular markers to investigate whether RA acts in anterior–posterior axial patterning in Oikopleura embryos. Results revealed that RA does not cause homeotic posteriorization in Oikopleura as it does in vertebrates and cephalochordates, and showed that a chordate can develop the phylotypic body plan in the absence of the classical morphogenetic role of RA. A comparison of Oikopleura and ascidian evidence suggests that the lack of RA-induced homeotic posteriorization is a shared derived feature of urochordates. We discuss possible relationships of altered roles of RA in urochordate development to genomic events, such as rupture of the Hox-cluster, in the context of a new understanding of chordate phylogeny
Evolution of developmental roles of Pax2/5/8 paralogs after independent duplication in urochordate and vertebrate lineages
Expression signatures of cisplatin- and trametinib-treated early-stage medaka melanomas
Small aquarium fish models provide useful systems not only for a better understanding of the molecular basis of many human diseases, but also for first-line screening to identify new drug candidates. For testing new chemical substances, current strategies mostly rely on easy to perform and efficient embryonic screens. Cancer, however, is a disease that develops mainly during juvenile and adult stage. Long-term treatment and the challenge to monitor changes in tumor phenotype make testing of large chemical libraries in juvenile and adult animals cost prohibitive. We hypothesized that changes in the gene expression profile should occur early during anti-tumor treatment, and the disease-associated transcriptional change should provide a reliable readout that can be utilized to evaluate drug-induced effects. For the current study, we used a previously established medaka melanoma model. As proof of principle, we showed that exposure of melanoma developing fish to the drugs cisplatin or trametinib, known cancer therapies, for a period of seven days is sufficient to detect treatment-induced changes in gene expression. By examining whole body transcriptome responses we provide a novel route toward gene panels that recapitulate anti-tumor outcomes thus allowing a screening of thousands of drugs using a whole-body vertebrate model. Our results suggest that using disease-associated transcriptional change to screen therapeutic molecules in small fish model is viable and may be applied to pre-clinical research and development stages in new drug discovery
Female Sex Development and Reproductive Duct Formation Depend on Wnt4a in Zebrafish.
In laboratory strains of zebrafish, sex determination occurs in the absence of a typical sex chromosome and it is not known what regulates the proportion of animals that develop as males or females. Many sex determination and gonad differentiation genes that act downstream of a sex chromosome are well conserved among vertebrates, but studies that test their contribution to this process have mostly been limited to mammalian models. In mammals, WNT4 is a signaling ligand that is essential for ovary and Müllerian duct development, where it antagonizes the male-promoting FGF9 signal. Wnt4 is well conserved across all vertebrates, but it is not known if Wnt4 plays a role in sex determination and/or the differentiation of sex organs in nonmammalian vertebrates. This question is especially interesting in teleosts, such as zebrafish, because they lack an Fgf9 ortholog. Here we show that wnt4a is the ortholog of mammalian Wnt4, and that wnt4b was present in the last common ancestor of humans and zebrafish, but was lost in mammals. We show that wnt4a loss-of-function mutants develop predominantly as males and conclude that wnt4a activity promotes female sex determination and/or differentiation in zebrafish. Additionally, both male and female wnt4a mutants are sterile due to defects in reproductive duct development. Together these results strongly argue that Wnt4a is a conserved regulator of female sex determination and reproductive duct development in mammalian and nonmammalian vertebrates
Aldh2 is a lineage-specific metabolic gatekeeper in melanocyte stem cells
Melanocyte stem cells (McSCs) in zebrafish serve as an on-demand source of melanocytes during growth and regeneration, but metabolic programs associated with their activation and regenerative processes are not well known. Here, using live imaging coupled with scRNA-sequencing, we discovered that, during regeneration, quiescent McSCs activate a dormant embryonic neural crest transcriptional program followed by an aldehyde dehydrogenase (Aldh) 2 metabolic switch to generate progeny. Unexpectedly, although ALDH2 is well known for its aldehyde-clearing mechanisms, we find that, in regenerating McSCs, Aldh2 activity is required to generate formate – the one-carbon (1C) building block for nucleotide biosynthesis – through formaldehyde metabolism. Consequently, we find that disrupting the 1C cycle with low doses of methotrexate causes melanocyte regeneration defects. In the absence of Aldh2, we find that purines are the metabolic end product sufficient for activated McSCs to generate progeny. Together, our work reveals McSCs undergo a two-step cell state transition during regeneration, and that the reaction products of Aldh2 enzymes have tissue-specific stem cell functions that meet metabolic demands in regeneration
Stacks: Building and Genotyping Loci De Novo From Short-Read Sequences
Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress. Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks can recover thousands of single nucleotide polymorphism (SNP) markers useful for the genetic analysis of crosses or populations. Stacks can generate markers for ultra-dense genetic linkage maps, facilitate the examination of population phylogeography, and help in reference genome assembly. We report here the algorithms implemented in Stacks and demonstrate their efficacy by constructing loci from simulated RAD-tags taken from the stickleback reference genome and by recapitulating and improving a genetic map of the zebrafish, Danio rerio
Sex Reversal in Zebrafish fancl Mutants is Caused by Tp53-Mediated Germ Cell Apoptosis
The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination
Retinoic Acid metabolic genes, meiosis, and gonadal sex differentiation in zebrafish.
To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish
Genome Evolution and Meiotic Maps by Massively Parallel DNA Sequencing: Spotted Gar, an Outgroup for the Teleost Genome Duplication
Genomic resources for hundreds of species of evolutionary, agricultural, economic, and medical importance are unavailable due to the expense of well-assembled genome sequences and difficulties with multigenerational studies. Teleost fish provide many models for human disease but possess anciently duplicated genomes that sometimes obfuscate connectivity. Genomic information representing a fish lineage that diverged before the teleost genome duplication (TGD) would provide an outgroup for exploring the mechanisms of evolution after whole-genome duplication. We exploited massively parallel DNA sequencing to develop meiotic maps with thrift and speed by genotyping F1 offspring of a single female and a single male spotted gar (Lepisosteus oculatus) collected directly from nature utilizing only polymorphisms existing in these two wild individuals. Using Stacks, software that automates the calling of genotypes from polymorphisms assayed by Illumina sequencing, we constructed a map containing 8406 markers. RNA-seq on two map-cross larvae provided a reference transcriptome that identified nearly 1000 mapped protein-coding markers and allowed genome-wide analysis of conserved synteny. Results showed that the gar lineage diverged from teleosts before the TGD and its genome is organized more similarly to that of humans than teleosts. Thus, spotted gar provides a critical link between medical models in teleost fish, to which gar is biologically similar, and humans, to which gar is genomically similar. Application of our F1 dense mapping strategy to species with no prior genome information promises to facilitate comparative genomics and provide a scaffold for ordering the numerous contigs arising from next generation genome sequencing
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