Project description:In birds and mammals, all mesoderm cells are generated from the primitive streak. Nascent mesoderm cells contain unique dorso-ventral (D/V) identities depending on their relative ingression position along the streak. Molecular mechanisms controlling this initial phase of mesoderm diversification are not well-understood. Using chick model, we generated high-quality transcriptomic datasets of different streak regions and analyzed their molecular heterogeneity. Streak tissues were dissected from stage-matched HH4 chick embryos and were further divided into four equal pieces along its anterio-posterior length (termed “A”, “B”, “C” and “D”, with “A” representing most anterior and therefore most dorsal and “D” most posterior and therefore most ventral). Each of the four regions was represented by three independent samples (n=3), with each sample containing 5-7 μg total RNA derived from 85-93 pooled pieces. 5 μg of RNA from each sample were used to screen Affymetrix Chicken Genome Array without an amplification step.
Project description:Transcriptional profiling of the preingression epiblast versus lateral epiblast, of preingression epiblast from control embryos treated with the DMSO carrier vehicle (0.5%) versus embryos treated with SU5402, U0126 or LY294002. Embryos were stages 5-6 at time of tissue isolation. The preingression epiblast (E2 region) is the region of epiblast just lateral to the primitive streak. Lateral epiblast is the E3 region. Two condition experiment. Samples for each condition isolated from at least 30 embryos. The experiments used isolated preingression epiblast, which is defined as the region of epiblast adjacent to the primitive streak that will ingress through the primitive streak. Preingression epiblast was isolated by microdissection using tungsten needles from control embryos or embryos treated with SU5402, U0126 or LY294002.
Project description:Sex determination is still poorly understood in birds and no key determinants have so far been identified. In contrast to most other species, dosage compensation of bird sex chromosomal genes appears rather ineffective. By comparing microarrays of microdissected primitive streak from single chicken embryos, we identified a large number of genes differentially expressed between male and female embryos at a very early stage (Hamburger and Hamilton stage 4), long before any sexual differentiation occurs. Most of these genes are located on the Z chromosome, which indicates that dosage compensation is ineffective in early chicken embryos. Gene ontology analyses using an enhanced annotation tool for Affymetrix probesets of the chicken genome shows that among the male-biased genes found on the Z chromosome, more than 20 genes have a role in sex differentiation. Experiment Overall Design: Primitive streak tissues were dissected out of individual Hamburger and Hamilton (HH) stage 4 chicken embryos and extracted for total RNA. Total RNA was amplified 2-cycles, biotin-labeled and hybridized to Affymetrix chicken GeneChip. Gene expression profiles of female and male samples were analyzed for sex-dimorphic expression.
Project description:Transcriptional profiling of the preingression epiblast versus lateral epiblast, of preingression epiblast from control embryos treated with the DMSO carrier vehicle (0.5%) versus embryos treated with SU5402, U0126 or LY294002. Embryos were stages 5-6 at time of tissue isolation. The preingression epiblast (E2 region) is the region of epiblast just lateral to the primitive streak. Lateral epiblast is the E3 region. Two condition experiment. Samples for each condition isolated from at least 30 embryos.
Project description:Abstract from Vermillion et al: During vertebrate development, progenitor cells give rise to tissues and organs through a complex choreography that commences at gastrulation. A hallmark event of gastrulation is the formation of the primitive streak, a linear assembly of cells along the anterior-posterior (AP) axis of the developing organism. To examine the primitive streak at a single-cell resolution, we measured the transcriptomes of individual chick cells from the streak or the surrounding tissue (the rest of the area pellucida) in Hamburger-Hamilton stage 4 embryos. The single-cell transcriptomes were then ordered by the statistical method Wave-Crest to deduce both the relative position along the AP axis and the prospective lineage of single cells. The ordered transcriptomes reveal intricate patterns of gene expression along the primitive streak.