Whole transcriptomic sequencing of zebrafish rx3 mutants during optic vesicle morphogenesis
ABSTRACT: To identify genes regulated by Rx3 during optic vesicle morphogenesis, adult zebrafish carriers of a null rx3 mutation were mated. Before 13 hours post fertilization (hpf), the earliest time point at which optic vesicle evagination phenotypes could be reliably detected, offspring were phenotypically separated into pools comprising of mutants with an absence of optic vesicles or siblings exhibiting a wild-type phenotype. Three replicates of pooled RNA samples from 13 hpf eyeless mutants (rx3-/-) or phenotypically wild-type siblings (rx3+/+ or rx3+/-), and one replicate of 13 hpf wild-type zebrafish larva were collected for whole transcriptome sequencing. Whole transcriptome sequencing (RNA-seq) was performed on zebrafish rx3-/- mutants, wild-type siblings and wild-type AB strains at 13 hpf
Project description:The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Teadresponsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosagedependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson’s chorioretinal atrophy and congenital retinal coloboma. 60 pooled eyes from 36 hpf wild type or vsx2:Gal4/dsRed:14xUAS:YapS87A embryos were pooled for one sample. Three wild type and three vsx2:Gal4/dsRed:14xUAS:YapS87A pools were analyzed for RNA.
Project description:We conducted functional analysis on zebrafish mutant fn10a by polyA+, 100bp paired end, strand-specific RNA-seq. By comparing the transcriptome of fn10a mutants and their wildtype siblings or wildtype controls, we revealed a transcriptome-wide RNA splicing deficiency and a large amount of intron-retaining transcripts, which resulted in compromised nonsense-mediated RNA decay and activation of the p53 pathway in fn10a mutants. 2 biological replicates were performed to consolidate the findings. We also conducted subcellular RNA-seq between fn10a mutants and wildtype siblings, to investigate the localization of intron retaining transcripts. Overall design: The wild-type control group (WT-control group) of 36-hpf embryos was generated from mating crosses between wild-type TL zebrafish, while fn10a mutants (mutant group) and siblings (WT-sibling group) were generated from mating crosses of fn10a heterozygote zebrafish. RNA-seq of 36hpf embryos for fn10a mutants, their wildtype siblings and wildtype TL. RNA-seq of cytoplasm RNA and nuclear RNA from 36hpf embryos for fn10a mutants and their wildtype siblings.
Project description:To investigate its role during zebrafish development, wnt signalling was either activated by the conditional expression of wnt8b or inhibited by the conditional expression of dickkopf-1b (dkk1b) under the control of the hsp70 heat shock promoter. The transgenic constructs contained a gfp reporter fusion to differentiate between transgenic embryos and their wild type siblings. Transgenic embryos and wild type control embryos at the 48 hours post fertilization stage (hpf) were exposed to heat shock (37 C) for 2 hours and were collected 4 hours after heat shock. The transcriptional response to activated or inhibited wnt signalling was profiled using microarrays and compared to wild type expression.
Project description:Bmp4 was conditionally deleted from the mouse optic vesicle with Rx-Cre. The heads of wild type and knockout E10.5 mouse embryos were laser micodissected to isolate the lens ectoderm and prospective retina from three embryos of each genotype. Total RNA was purified and reverse transcribed and amplified using a NuGEN Ovation Pico WTA system V2 kit. cDNA was biotinylated and hybridized to Illumina Mouse6 V2 bead arrays. Three wild type and three knockout embryos were used. RNA obtained from the lens ectoderm or prospective retina (distal optic vesicle) was pooled, amplified and used for microarray analysis. Statistical analysis was performed using the values from the 25-50 beads on each array.
Project description:To determine the molecular basis for the reduced blood cells in circulation in ezh2 mutant zebrafish, we performed high-throughput sequencing at 28 hpf. Results revealed 893 up-regulated genes and 425 down-regulated genes in the ezh2 mutant zebrafish compared with wild-type zebrafish. We found that some hematopoietic genes are down-regulated in ezh2 mutant zebrafish, which was reconfirmed with qRT-PCR. Overall design: mRNA profiles of 28 hpf wild-type and ezh2 mutant zebrafish were generated by deep sequncing.
Project description:Goal of this study is differential gene expression between wild type and Toddler mutant during early zebrafish embryogenesis Overall design: Four timepoints - 4 hours post fertilization (hpf), 5 hpf, 6 hpf, and 7 hpf; one replicate of wild type at each time point, one replicate Toddler mutant at each time point
Project description:In order to discover the targets of Foxj1, we made transgenic zebrafish in which Foxj1 is ubiquitously overexpressed in response to heat [Tg(hsp70::foxj1a)]. Transgenic embryos and wild type control embryos were collected, given two heat shocks (at 18 hours post fertilization (hpf) and 20 hpf), then analyzed at 22 hpf. Gene expression profiles of embryos overexpressing Foxj1a were compared to gene expression profiles of wild type embryos using Nimblegen whole transcriptome zebrafish microarrays.
Project description:Goal of this study is differential gene expression between wild type and MZnanog mutant during early zebrafish embryogenesis Overall design: Three timepoints - 2 hours post fertilization (hpf), 4 hpf, and 6.5 hpf; two replicates of wild type at each time point, one replicate for MZnanog at each time point
Project description:Eye formation is regulated by a complex network of eye field transcription factors (EFTFs), including LIM-homeodomain gene LHX2. We disrupted LHX2 function at different stages during this process using a conditional knock-out strategy in mice. We find that LHX2 function is required in an ongoing fashion to maintain optic identity across multiple stages, from the formation of the optic vesicle to the differentiation of the neuroretina. At each stage, loss of Lhx2 led to upregulation of a set of molecular markers that are normally expressed in the thalamic eminence and in the anterodorsal hypothalamus in a portion of the optic vesicle or retina. Furthermore, the longer LHX2 function was maintained, the further optic morphogenesis progressed. Early loss of function caused profound mispatterning of the entire telencephalic-optic-hypothalamic field, such that the optic vesicle became mispositioned and appeared to arise from the diencephalic-telencephalic boundary. At subsequent stages, loss of Lhx2 did not affect optic vesicle position but caused arrest of optic cup formation. If Lhx2 was selectively disrupted in the neuroretina from E11.5, the neuroretina showed gross dysmorphology along with aberrant expression of markers specific to the thalamic eminence and anterodorsal hypothalamus. Our findings indicate a continual requirement for LHX2 throughout the early stages of optic development, not only to maintain optic identity by suppressing alternative fates but also to mediate multiple steps of optic morphogenesis. These findings provide new insight into the anophthalmic phenotype of the Lhx2 mutant and reveal novel roles for this transcription factor in eye development. Overall design: Neuroretina from embryonic day 13.5 mice from ;Lhx2 lox/lox and Chx10-Cre;Lhx2 lox/lox mice were dissescted and analyzed. Two replicates for each genotype were tested.