Project description:The BMP signaling pathway regulates multiple steps of hematopoiesis, mediated through receptor-regulated Smads, including Smad1 and Smad5. Here we use loss-of-function approaches in zebrafish to compare the roles of Smad1 and Smad5 during embryonic hematopoiesis. Microarray experiments revealed that the two proteins regulate redundantly the key initiators of the hemato-vascular program, including scl, lmo2, and gfi1. However, each also regulates a remarkably distinct genetic program, with Smad5 uniquely regulating the BMP signaling pathway itself. Our results suggest that specificity of BMP signaling output, with respect to hematopoiesis, can be explained by differential functions of Smad1 and Smad5. Keywords: Gene expression transcript profiles
Project description:The BMP signaling pathway regulates multiple steps of hematopoiesis, mediated through receptor-regulated Smads, including Smad1 and Smad5. Here we use loss-of-function approaches in zebrafish to compare the roles of Smad1 and Smad5 during embryonic hematopoiesis. Microarray experiments revealed that the two proteins regulate redundantly the key initiators of the hemato-vascular program, including scl, lmo2, and gfi1. However, each also regulates a remarkably distinct genetic program, with Smad5 uniquely regulating the BMP signaling pathway itself. Our results suggest that specificity of BMP signaling output, with respect to hematopoiesis, can be explained by differential functions of Smad1 and Smad5. Keywords: Gene expression transcript profiles The experiment was designed to identify the unique Smad1 and Smad5 dependent transcripts during the somitogenesis stage of development, during which mesoderm is specified to the hematopoietic lineage. Embryos were injected with translational blocking morpholinos for Smad1, Smad5 or both, and then collected at the 1-somite stage for RNA extraction. For every experiment control uninjected wildtype sibling embryo were also collected for comparison. Three biological replicates were done for each knockdown set. Total RNA was sent to Nimblegen for cDNA synthesis, dye labeling and hybridization. Single knockdown samples were hybridized to the Nimblegen 2006 Danio rerio Gene Expression Array chip and the double knockdown samples to the 2007 verison of the chip, which contains the same test genes, but with additional control oligos. Dye swaps were done for each set; for 2 of the 3 hybridization in each set Cy3 was the dye used for the experimental sample and in the 3rd Cy5 was used. The raw hybridization data was obtained from Nimblegen, normalized using NimbleScan and anaylzed using R software.
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: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).