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| Status |
Public on Sep 20, 2006 |
| Title |
Time course microarray approach comparing the ectopic expression and loss-of-function of gcm to wildtype |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by array
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| Summary |
The determination of glial fate in the developing embryonic nervous system of Drosophila is dependent on the master regulatory gene glial cells missing (gcm). gcm encodes a transcription factor and serves as a binary switch by inducing the glial fate and repressing the neuronal pathway. The ectopic expression of gcm throughout the CNS leads to excessive glia on the cost of presumptive neurons, whereas gcm loss-of-function embryos lack nearly all lateral glial cells. To date only little is known about the genetic program underlying glial differentiation.
In order to identify glial specific genes downstream of gcm we performed a whole-genome microarray approach, where we compared the gcm gain-of-function situation (GOF) and, for the first time, the gcm loss-of-function (LOF) against wildtype in a time course experiment throughout embryogenesis. Intense filtering of differentially regulated genes on behalf of statistics as well as developmental means such as profile of differential regulation enabled us to identify more than 40 novel glial genes. The confirmation of these genes by in situ hybridization revealed temporal expression profiles in all or subsets of glial cells. We give a detailed description of the expression patterns of the candidate genes and adress their regulation in the glial pathway in different glial mutant backgrounds. Additionally we started to analyze mutant phenotypes of candidate genes to clarify their functional relevance for glial differentiation.
Keywords: Drosophila,gliogenesis,time course,loss-of-function,gain-of-function
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| Overall design |
Cy5/Cy3 labeled RNA was co-hybridized onto a QMT Amino (Quantifoil) glass slide. For wt control we pooled mRNA from UAS-gcm stock B5446 and Gal4-line MZ1060 in equal amounts.
Sample collection:
Embryo samples from the MZ1060 x B5446, B5446 and MZ1060 were collected in parallel as one-hour egg lays at 25°C, which were allowed to develop to the desired stage at 29°C/18°C.
gcmN7-4 mutant flies were balanced with either Kr-GFP (for stages 10 to 13) or Ubi-GFP (for stages 14 to 16) balancer chromosomes. Eggs were collected as described above, transferred into PBS buffer supplemented with 0.3% Tween 20 and homozygous mutant embryos were automatically sorted with the CopasTM Select embryo sorter (Union Biometrica, Somerville, MA, USA) by their lack of GFP-expression.
First total RNA was isolated from all samples using the Rneasy Kit and then mRNA was isolated using the Oligotex Kit (both Qiagen,Hilden,Germany)
Microarray hybridization:
At least four independent experiments were performed for each of the 8 different stages
(embryonic stages 9 to 16) in competitive hybridizations against the pooled wildtype control of the same stage.
At least one dye swap was included in each of the repetitions.
We used the indirect labeling method, with 4,5µg random hexamer primer and 0,75µg Poly-dT primer ( both Invitrogen, Karlsruhe, Germany) added to 1µg mRNA.
For hybridization, the labeled cDNAs were taken up in SlideHyb buffer #1 (Ambion, Woodward, USA), denatured at 95°C for 5min,
and applied to the array. Hybridization was performed in appropriate chambers (TeleChem) in a waterbath at 55°C over night.
The hybridized glass slides were scanned on a ScanArray 5000 (Perkin Elmer, Wellesley, USA) using the ScanArray software (Version 3.1).
The resulting images (TIFF format, 16-bit grayscale) for each channel - Cy5 (632nm) and Cy3 (532nm) - were analyzed
further with the GenePix software (Version 5.0; Axon Instruments, Union City, USA).
As each microarray contains two replicates for each gene, we used the standard deviation (SD)
of their dye-ratio (Cy5/Cy3) to filter for reproducible hybridizations. Only if at least 30% of all genes on the array
had a SD of log(632/532) of less than 1/3 between the replicates was the microarray included for further analysis.
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| Contributor(s) |
Altenhein B, Becker A, Beckmann B, Busold C, Hoheisel J, Technau GM |
| Citation(s) |
16762338 |
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| Submission date |
Sep 07, 2005 |
| Last update date |
Mar 16, 2012 |
| Contact name |
Angela Becker |
| E-mail(s) |
[email protected]
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| Organization name |
University of Mainz
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| Department |
Institute of Genetics
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| Lab |
AG Technau
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| Street address |
Becherweg 32
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| City |
Mainz |
| ZIP/Postal code |
55099 |
| Country |
Germany |
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| Platforms (1) |
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| Samples (58)
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| Relations |
| BioProject |
PRJNA92909 |
| Supplementary data files not provided |
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