Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Targeting the hemangioblast with a novel cell type-specific enhancer

ABSTRACT: Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker. We report the identification of a hemangioblast-specific enhancer (Hb) located in the cis-regulatory region of chick Cerberus gene (cCer) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis. We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development. We used microarray analysis of Hb-eGFP expressing cells to verify the expression of hemangioblast-specific genes in this cell population. In order to characterize the gene expression profile of the Hb-eGFP-positive population, we have isolated Hb-eGFP-positive cells and compared their expression profile with the control RFP-positive population. In brief, chicken embryos were electroporated at HH3 with Hb-eGFP and pCAGGS-RFP reporter constructs, observed under a fluorescence stereoscope and harvested at stage HH5-6 into three groups of four embryos each. Embryos were dissociated into single cell suspensions and the eGFP-positive and eGFP-negative/RFP-positive cell populations were sorted by Fluorescence Activated Cell Sorting (FACS). Cell populations were collected simultaneously into two different tubes, containing RNAlater (Ambion) and subsequently used for RNA extraction. Total RNA was isolated from triplicates of each population, evaluated for RNA integrity, reverse transcribed, amplified and hybridized against six Affymetrix Chicken Genome microarrays.

ORGANISM(S): Gallus gallus

SUBMITTER: Ana Tavares

PROVIDER: E-GEOD-32494 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Genetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their other targets and the pathways they constitute, remain largely unknown. In the avian embryo, Cerberus (cCer) transcripts are detected in the anterior mesendoderm including the heart precursor cells and in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression into the heart and the hemangioblast precursor cells, allowing us to identify a population of cells that consistently exit from the anterior primitive streak region from as early as stage HH3+ and that later will populate the heart. In order to identify and study novel genes expressed and involved in the correct development and differentiation of the vertebrate H/HPC (Heart/ Hemangioblast Precursor Cells) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 800 transcripts potentially expressed in these precursor lineages. We have identified unknown genes that are differentially expressed in the H/HPC precursor lineages. By developing a procedure to isolate the heart precursor cells using the cer2.5-EGFP construct, we were able to specifically isolate a population of H/HPC expressing already known cardiac markers, and a long list of still uncharacterized genes. From those, we defined by WISH that at least some of these in silico identified transcripts are in fact expressed in these cell populations and more importantly, functionally required for heart formation.More importantly, our study unveiled several uncharacterized genes that can now be used for further studies. Portions of chick embryos were selected and excised at early stages of development for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of embryos at early gastrula stage in order to increase the temporal resolution of expression profiles. To that end, we hand-selected and excised portions of the embryos according to morphological criteria and EGFP expression. Four GeneChip chicken arrays used for another study were added to the two arrays in this study to ensure a robust model for expression value computation (Note: The 4 samples chick_T2-1, chick_T2-2, chick_T2-3, chick_T2-4, were performed by another group and for a different array, and nothing has to do with our data. They were only run at the same time and used for normalization. Raw data unavailable.). The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method. Normalized CEL intensities of the six arrays were used to obtain model-based gene expression indices (MBEI) based on a PM (Perfect Match)-only model. Processed data for all 6 samples can be found in supplementary file linked below.

Project description:The MTF-1 transcription factor is considered to be a master regulator of zinc homoestasis. We previously characterized a constitutively nuclear, dominant-negative zebrafish MTF-1/eGFP fusion protein (dnMTF-1). In this study, in vitro transcribed dnMTF-1 mRNA was microinjected into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. The role of MTF-1 signaling in zebrafish embryos was examined by injection of in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. A Narishige IM-300 microinjector was used to microinject 1-2 cell embryos with ~100 pg of dnMTF-1 IVT mRNA (~2.1 nL microinjection volume with a concentration 50 ng/M-BM-5L IVT mRNA), or ~100 pg of enhanced green fluorescent protein (eGFP) IVT mRNA. Immediately after microinjection, embryos from each treatment were divided into 4 replicates of 30 pooled embryos and held in 25 mL of 0.3X DanieauM-bM-^@M-^Ys at 28.5M-BM-0C under a 14 hour light/10 hour dark cycle. eGFP positive embryos (dnMTF-1 and eGFP) were collected at 28 and 36 hpf and flash frozen in liquid nitrogen for future RNA isolation and microarray hybridization. Total RNA was prepared using the RNA STAT60 protocol (Tel-Test, Inc., TX). RNA was quantified using a Nanodrop 2000C spectrophotometer and assessed for quality using an Agilent 2100 Bioanalyzer Lab-on-chip system. Only samples with RNA integrity number (RIN) between 9.8-10 were used for microarray analysis. For each RNA sample, a single microarray was hybridized with 750 ng Cy3 labeled cDNA using AgilentM-bM-^@M-^Ys standard conditions for single-color microarrays. The samples were hybridized to a the Agilent 4x44K feature zebrafish microarray using the Agilent In situ Hybridization Kit Plus by the Genome Technology Core at the Whitehead Institute for Biomedical Research (Cambridge, MA). Post-hybridization, microarray slides were washed as per the Agilent In situ Hybridization Kit Plus and scanned with an Agilent DNA Microarray Scanner. Extraction of raw microarray results was performed using AgilentM-bM-^@M-^Ys Feature Extraction software with background detrending (spatial and multiplicative). The data were normalized using a non-linear scaling method based on rank invariant probes. Cy3 signal saturated probes and probes not above background in all replicated were then removed prior to statistical analysis. Statistical tests were performed using the MeV microarray analysis suite. All data were log transformed and median centered for each probe. A two-factor ANOVA was performed for time (28 vs. 36 hpf), treatment (dnMTF-1 vs. eGPF), and their interaction with p-value based on 1000 permutations of the data and alpha of 0.01. For post-hoc analyses to find biologically relevant results, the probes significant for treatment and time-treatment interaction were secondarily examined using Rank Product (RP) analysis. Rank product analysis was performed to provide lists of probes up- and down-regulated between GFP control and MTF-1 for each time point. For each test, a two-class unpaired RP analysis was performed using 1000 permutation of the data with a false discovery rate (FDR) not exceeding 5%.

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Targeting the hemangioblast with a novel cell type-specific enhancer

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