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Series GSE28441 Query DataSets for GSE28441
Status Public on May 27, 2011
Title Transcriptional profiling of fetal hypothalamic TRH neurons
Organism Rattus norvegicus
Experiment type Expression profiling by array
Summary During murine hypothalamic development, different neuroendocrine cell phenotypes are generated in overlapping periods; this suggests that cell-type specific developmental programs operate to achieve complete maturation. A balance between programs that include cell proliferation, cell cycle withdrawal as well as epigenetic regulation of gene expression characterizes neurogenesis. Thyrotropin releasing hormone (TRH) is a peptide that regulates energy homeostasis and autonomic responses. To better understand the molecular mechanisms underlying TRH neuron development, we performed a genome wide study of its transcriptome during fetal hypothalamic development.
In primary cultures, TRH cells constitute 2% of the total fetal hypothalamic cell population. To purify these cells, we took advantage of the fact that the segment spanning –774 to +84 bp of the Trh gene regulatory region confers specific expression of the green fluorescent protein (GFP) in the TRH cells. Transfected TRH cells were purified by fluorescence activated cell sorting, various cell preparations pooled, and their transcriptome compared to that of GFP- hypothalamic cells. TRH cells undergoing the terminal phase of differentiation, expressed genes implicated in protein biosynthesis, intracellular signaling and transcriptional control. Among the transcription-associated transcripts, we identified the transcription factors Klf4, Klf10 and Atf3, which were previously uncharacterized within the hypothalamus.
To our knowledge, this is the first report identifying transcripts with a potentially important role during the development of a specific hypothalamic neuronal phenotype. This genome-scale study forms a rational foundation for identifying genes that might participate in the development and function of hypothalamic TRH neurons.
 
Overall design Hypothalamic primary cultures were prepared from E17 rat embryos. Twenty-four hours after seeding, cells were transfected using the minimal Trh promoter (-776/+84 bp) upstream of the GFP. Forty-eight hours after transfection, cells were trypsinized and subjected to FACS. TRH+ cells were purified from a pool of five 60-mm dishes using the FACS Vantage (Becton Dickinson, San Jose, CA) and the exclusion method at high speed (60 µl/min). In general, 20,000 GFP+ cells were purified from 5 X 106 cells. The microarray analysis was performed as described in the Affymetrix expression analysis technical manual (http://www.affymetrix.com). Total RNA (10 µg) was extracted from three different cell populations: i) sorted TRH-GFP+ cells (GFP+); ii) TRH-GFP+ and GFP- mixed cells (GFP+/-) passed through the FACS but not sorted, and iii) non transfected cells (NT). To obtain a sufficient amount of RNA for each cell population, the number of independent experiments pooled for the GFP+ sample was higher than for the other samples. Therefore, a pool of six independent experiments was used to prepare total RNA from the GFP+ and three independent experiments for GFP+/- or NT cells.
 
Contributor(s) Guerra-Crespo M, Pérez-Monter C, Chandra Janga S, Castillo-Ramírez S, Gutierrez-Rios RM, Joseph-Bravo P, Pérez-Martínez L, Charli J
Citation(s) 21569245
Submission date Apr 07, 2011
Last update date Feb 21, 2017
Contact name Leonor Perez-Martinez
E-mail(s) leonor@ibt.unam.mx
Phone 52-777-329-1600
Fax 52-777-317-2388
Organization name University of Mexico
Department Medicina Molecular y Bioprocesos
Lab Neuroimmunobiology
Street address Avenida Universidad 2001
City Cuernavaca
State/province Morelos
ZIP/Postal code 62271
Country Mexico
 
Platforms (1)
GPL85 [RG_U34A] Affymetrix Rat Genome U34 Array
Samples (5)
GSM703090 Sorted TRH, GFP+, biological rep 1
GSM703091 Sorted TRH, GFP+, biological rep 2
GSM703092 Total Cell Population, GFP +/-, biological rep 1
Relations
BioProject PRJNA139251

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE28441_RAW.tar 24.4 Mb (http)(custom) TAR (of CEL, CHP)
Processed data included within Sample table
Processed data provided as supplementary file

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