Evaluation of gene expression profiles and pathways underlying postnatal development in mouse sclera.
OBJECTIVE
The aim of this study was to identify the genes and pathways underlying the growth of the mouse sclera during postnatal development.


METHODS
Total RNA was isolated from each of 30 single mouse sclera (n=30, 6 sclera each from 1-, 2-, 3-, 6-, and 8-week-old mice) and reverse-transcribed into cDNA using a T7-N(6) primer. The resulting cDNA was fragmented, labeled with biotin, and hybridized to a Mouse Gene 1.0 ST Array. ANOVA analysis was then performed using Partek Genomic Suite 6.5 beta and differentially expressed transcript clusters were filtered based on a selection criterion of ≥ 2 relative fold change at a false discovery rate of ≤ 5%. Genes identified as involved in the main biologic processes during postnatal scleral development were further confirmed using qPCR. A possible pathway that contributes to the postnatal development of the sclera was investigated using Ingenuity Pathway Analysis software.


RESULTS
The hierarchical clustering of all time points showed that they did not cluster according to age. The highest number of differentially expressed transcript clusters was found when week 1 and week 2 old scleral tissues were compared. The peroxisome proliferator- activated receptor gamma coactivator 1-alpha (Ppargc1a) gene was found to be involved in the networks generated using Ingenuity Pathway Studio (IPA) from the differentially expressed transcript cluster lists of week 2 versus 1, week 3 versus 2, week 6 versus 3, and week 8 versus 6. The gene expression of Ppargc1a varied during scleral growth from week 1 to 2, week 2 to 3, week 3 to 6, and week 6 to 8 and was found to interact with a different set of genes at different scleral growth stages. Therefore, this indicated that Ppargc1a might play a role in scleral growth during postnatal weeks 1 to 8.


CONCLUSIONS
Gene expression of eye diseases should be studied as early as postnatal weeks 1-2 to ensure that any changes in gene expression pattern during disease development are detected. In addition, we propose that Ppargc1a might play a role in regulating postnatal scleral development by interacting with a different set of genes at different scleral growth stages.
Singapore Eye Research Institute, Singapore.
Wan'E
W
Lim
Jia Lin
JL
Kwan
Liang Kee
LK
Goh
Roger W
RW
Beuerman
Veluchamy A
VA
Barathi
D000818:Animals; D000831:Animals, Newborn; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D053263:Gene Regulatory Networks; D016678:Genome; D051379:Mice; D008807:Mice, Inbred BALB C; D005810:Multigene Family; D020411:Oligonucleotide Array Sequence Analysis; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000254:growth & development / Q000378:metabolism*; D012984:Software; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors
D005136:Eye Proteins; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C485290:Ppargc1a protein, mouse; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors