Abstract
Purpose: :
Pitx3 is a homeodomain transcription factor that is expressed during lens development and is associated with lens degeneration/cataracts when mutated in vertebrates. In order to identify downstream targets of PITX3, PITX3 was both overexpressed and downregulated in human lens epithelial cells and transcriptomes from these preparations were analyzed using microarray assays. Expression of GREM1 was found to be downregulated in cells overexpressing PITX3 and upregulated in cells with lower expression of PITX3. These results were verified by quantitative real time PCR. In order to investigate whether gremlin is involved in the regulation of lens development by pitx3 in vivo, we performed further studies in zebrafish pitx3 morphants.
Methods: :
In situ hybridization was used to examine the expression patterns of gremlin1 and gremlin2 in wild-type zebrafish embryos as well as pitx3 morphants at different stages of development. We examined grem1 mutants to identify gremlin mutant phenotype.
Results: :
In wild-type embryos, grem1 was found to be initially expressed in the lens and developing ganglion cells at 24 hpf. At 48 hpf, grem1 was detected in the ganglion cells but not in the lens. At 72 hpf, grem1 was downregulated in the lens and the ganglion cells and appeared to be present in the developing iris and cornea. In terms of grem2, in wild-type embryos it was found to be expressed in the choroid fissure at 24 hpf and in the ventral retina at 48 hpf; grem2 is not detected in the lens at any stage. In the pitx3 morphant embryos, grem1 but not grem2 expression was detected in the lens of 48 hpf and 72 hpf embryos.
Conclusions: :
Our results show that grem1 but not grem2 may be involved in the pitx3 regulation of lens development. Experiments are currently underway aimed at over/misexpression of grem1 in the developing lens in zebrafish using lens-specific crystallin promoter. These experiments will demonstrate whether misexpression of grem1 in the absence of pitx3 plays a role in lens degeneration phenotype associated with pitx3 deficiency.
Keywords: development • transcription factors • gene/expression