Project description:Classical embryological studies revealed that during mid-embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses, which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage, however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum, including a segmented worm, a flatworm, a roundworm, a water bear, a fruitfly, a sea urchin, a zebrafish, a sea anemone, a sponge, and a comb jelly. For each species, we collected the embryonic transcriptomes at ~100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly, we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation, and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes, also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility.
Project description:This project aimed at identifying developmental stage specific transcript profiles for catecholaminergic neurons in embryos and early larvae of zebrafish (Danio rerio). Catecholaminergic neurons were labeled using transgenic zebrafish strains to drive expression of GFP. At stages 24, 36, 72 and 96 hrs post fertilization, embryos were dissociated and GFP expressing cells sorted by FACS. Isolated RNAs were processed using either polyA selection and libray generation or NanoCAGE. This is the first effort to determine stage specific mRNA profiles of catecholaminergic neurons in zebrafish.
Project description:Classical embryological studies revealed that during mid-embryogenesis vertebrates show similar morphologies. This âphylotypic stageâ has recently received support from transcriptome analyses, which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a âzootypicâ stage, however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum, including a segmented worm, a flatworm, a roundworm, a water bear, a fruitfly, a sea urchin, a zebrafish, a sea anemone, a sponge, and a comb jelly. For each species, we collected the embryonic transcriptomes at ~100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly, we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation, and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes, also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. 106 single embryo samples
Project description:This project aimed at identifying developmental stage specific transcript profiles for catecholaminergic neurons in embryos and early larvae of zebrafish (Danio rerio). Catecholaminergic neurons were labeled using transgenic zebrafish strains to drive expression of GFP. At stages 24, 36, 72 and 96 hrs post fertilization, embryos were dissociated and GFP expressing cells sorted by FACS. Isolated RNAs were processed using either polyA selection and libray generation or NanoCAGE. This is the first effort to determine stage specific mRNA profiles of catecholaminergic neurons in zebrafish. Catecholaminergic neurons were labeled by four different strategies: (1) 24 hrs old embryos: we used the ETvmat2:GFP transgenic line (Wen et al. 2007). Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev Biol. 2008 Vol 314 p84-92) which at this early stage labels catecholaminergic neurons in posterior tuberculum and locus coeruleus; (2) 24 hrs old embryos: we used Tg(otpb.A:egfp)zc48 transgenic line (Fujimoto et al. Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification. Dev Biol 2011, Vol 352, p393–404) which at this stage label ventral diencephalic dopaminergic neurons and some preoptic neurons. (3) For 72 and 96 hrs old zebrafish larvae we used a th:GFP BAC transgenic lines that labels catecholaminergic neurons (Tay et al., Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat Comms 2011 Vol 2, 171; also: T. Leng and W. Driever, unpublished). (4) for the 36 and 48 hrs old zebrafish larvae we used a th:Gal4VP16 driver and UAS:EGFP responder transgenic line system to label catecholaminergic cells (Fernandes et al., Deep brain photoreceptors control light-seeking behavior in zebrafish larvae. Curr Biol. 2012 Vol 22 DOI 10.1016/j.cub.2012.08.016). We used the different transgenic lines, because lines (3) and (4) do not efficiently label catecholaminergic neurons at early stages, while lines (1) and (2) also have GFP expression in several other non-catecholaminergic populations at later stages of development. Embryos were dissociated and catecholaminergic neurons were FACS sorted from GFP-tagged zebrafish (Manoli and Driever, 2012, Cold Spring Harbor Protoc. DOI 10.1101/pdb.prot069633). RNA was either processed for NanoCAGE, or mRNA was isolated and amplified. cDNA was sequenced by Illumina technique. This data submission is a series of data files consisting of three independent experiments with diffrent RNA-Seq depth: Samples 1-4 (NanoCage): Samples 5-8 (RNA-Seq high read numbers), and SAmples 9-12 (RNA-Seq low read numbers).
Project description:Humans and animals have problems producing eggs with high embryo developmental competence, but the causes of poor egg quality are usually unknown. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading model for vertebrate development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were used to create pooled or replicated sample sets subjected to different levels of fractionation before LC-MS/MS. Obtained spectra were searched against a custom zebrafish proteome database and detected proteins were annotated, categorized and quantified based on their normalized spectral counts. Manual and automated enrichment analyses were highly confirmative, showing that good and poor quality eggs have disparate proteomes. Proteins involved in protein synthesis, energy metabolism, and lipid metabolism, and certain vitellogenin products were strikingly underrepresented in poor quality eggs. Poor quality eggs also had significantly higher representation of proteins related to immune system and endosome/lysosome functioning, oncogenes, and apoptosis, as well as lectins and egg envelope proteins. Quantitative comparisons of highly abundant proteins revealed 9 candidate egg quality markers warranting further study. In conclusion, the zebrafish egg proteome appears to be linked to embryo developmental potential, a phenomenon that begs further investigation.
Project description:Humans and animals have problems producing eggs with high embryo developmental competence, but the causes of poor egg quality are usually unknown. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading model for vertebrate development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were used to create pooled or replicated sample sets subjected to different levels of fractionation before LC-MS/MS. Obtained spectra were searched against a custom zebrafish proteome database and detected proteins were annotated, categorized and quantified based on their normalized spectral counts. Manual and automated enrichment analyses were highly confirmative, showing that good and poor quality eggs have disparate proteomes. Proteins involved in protein synthesis, energy metabolism, and lipid metabolism, and certain vitellogenin products were strikingly underrepresented in poor quality eggs. Poor quality eggs also had significantly higher representation of proteins related to immune system and endosome/lysosome functioning, oncogenes, and apoptosis, as well as lectins and egg envelope proteins. Quantitative comparisons of highly abundant proteins revealed 9 candidate egg quality markers warranting further study. In conclusion, the zebrafish egg proteome appears to be linked to embryo developmental potential, a phenomenon that begs further investigation.