Project description:We report differential transcriptomes of zebrafish embryos at 6 and 10 hours post fertilization. Specifically, we note a dramatic shift in the expression of genes involved in cellular metabolism from 6 to 10 hours post fertilization. This result may explain at least in part why metabolic demand of developing embryos at the examined stage is sharply increased.
Project description:Gastrulation is a milestone event of embryonic development, during which germ layers are specified and reassigned into the body plan. Due to the inaccessibility in vivo at this stage and the ethical limitations, gastrulation currently remains a mystery in primates. Here, we report the establishment of an in vitro system to culture cynomolgus monkey embryos after up to 20 days to develop beyond the initiation of gastrulation. The histology, immunostaining and single cell sequencing results showed that the in vitro cultured embryos largely recapitulated the key events of early post-implantation development, and initiated gastrulation in primates. Considering the high similarities between monkeys and humans, this system will provide a unique platform for the investigations of human early post-implantation development at physiological, pathological and environmental conditions.
Project description:Current knowledge of the mechanisms of cell migration is based on differentiated cells in culture where it is known that the actomyosin machinery drives migration via dynamic interactions with the extracellular matrix and adhesion complexes. However, unlike differentiated cells, cells in early metazoan embryos also dynamically change cell sizes as they migrate. The relevance of cell size to cell migration and embryonic development is not known. Here we investigate these phenomena in zebrafish embryos, a model system in which reductive cell divisions causes cell sizes to decrease naturally over time as cells migrate collectively to sculpt the embryonic body plan. Because mutations that can perturb cell sizes so early in development do not exist, we generate haploid and tetraploid zebrafish embryos and show that cell sizes in such embryos are smaller and larger than the diploid norm, respectively. Cells in embryos made of smaller or larger than normal cells migrate sub-optimally, leading to gastrulation defects. Multiple lines of evidence suggest that the observed defects originate from altered cell size, and not from pleotropic effects of altered ploidy. This interpretation is strengthened by finding that gastrulation defects are rescued by increasing cell sizes in embryos wherein cell sizes are smaller than normal. We show that the migration defects are cell-autonomous by live imaging migrating haploid and tetraploid cells during gastrulation in chimeric diploid embryos. Analysis of membrane protrusion dynamics in single cells shows that cells normally extend protrusions non-uniformly during migration, a phenomenon which is perturbed when cell sizes deviate from the norm. Thus, an optimal range of developmental stage-specific cell sizes appears necessary for collective cell migration to correctly position cells in space and time to shape an amorphous ball of blastoderm into an embryo.
Project description:Craniofacial and CNS malformations characteristic of Fetal Alcohol Syndrome are caused by alcohol exposure during gastrulation (embryonic day [E]7 in mice; 2nd – 3rd week of human pregnancy). Genetics are a known contributor to differences in alcohol sensitivity in humans, and between different animal model strains such as C57BL/6J and C57BL/6NHsd mice. To investigate how these two genetically similar mouse strains differ in alcohol sensitivity, we profiled gene expression at baseline (E7.0) and 6 or 12 hours after alcohol exposure (E7.25–E7.5) in gastrulation-stage embryos. Many of the baseline transcriptional differences across strains were associated with immune signaling, indicative of their molecular divergence. Alcohol exposure was associated with a more pronounced transcriptional effect in 6J than in 6N, matching the 6J’s increased sensitivity. Alcohol exposure upregulated pathways related to cell death, stress, and hypoxia, and downregulated proliferation and morphogenic pathways. Dysregulated genes were also associated with craniofacial and CNS defects, but only in the 6J embryos. These data demonstrate the genetic variation that can contribute to the effects of prenatal alcohol and identify impacted molecular pathways, including higher baseline expression of inflammatory signaling genes in 6J embryos that likely increases sensitivity to teratogens such as alcohol. Collectively, our data provide a valuable resource that not only enables the discovery of biomarkers pertinent to prenatal alcohol exposure, but also facilitates the transcriptional comparison of two genetically similar mouse strains at three finely resolved developmental timepoints. To this end, we developed a web-based interactive tool to aid in data exploration available at http://parnell-lab.med.unc.edu/Embryo-Transcriptomics/.
Project description:Transcriptional profiling of Mtx2 morphants at 30% epiboly stage, shield stage and tailbud stage of embryonic development: Coordinated cell movements of gastrulation reshape the uniform blastula into a structured gastrula with three fundamental axes and an internal germ layer of mesoderm. Genetic regulation of the forces which drive gastrulation is incompletely understood. We employed a microarray screen in zebrafish for genes which participate in epibloy, and identified many with biochemical roles in endocytosis, microtubule remodeling, cell adhesion and transcription.
Project description:Migrasomes are recently identified vesicular organelles that form on retraction fibers behind migrating cells, cellular contents are released from migrasome by a process named migracytosis. The function of migrasomes in living organisms is unknown. Here we show that migrasomes are formed during zebrafish gastrulation, signaling molecules such as chemokines are enriched in migrasomes. Migrasomes are clustered on spatially restricted area in embryo where they provide regional cues for organ morphogenesis. Our study shown migrasome is signaling organelles which integrate spatial and specific biochemical information to coordinate migrating cells in complex biological processes such as morphogenesis.
Project description:Here we describe successful implementation of CUT&RUN for profiling protein-DNA interactions in zebrafish embryos. We apply modified a CUT&RUN method to generate high resolution maps of enrichment for H3K4me3, H3K27me3, H3K9me3, and RNA polymerase II during zebrafish gastrulation. Using this data, we identify a conserved subset of developmental genes that are enriched in both H3K4me3 and H3K27me3 during gastrulation, and we demonstrate the increased effectiveness of CUT&RUN for detecting protein enrichment at repetitive sequences with reduced mappability. Our work demonstrates the power of combining CUT&RUN with the strengths of the zebrafish system to better understand the changing embryonic chromatin landscape and its roles in shaping development.