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L. E. Kelly, R. I. Martinez-de Luna, H. E. Moose, H. M. El-Hodiri; Involvement of Forkhead Transcription Factors in Progenitor Cell Biology of Xenopus Retinal Development and Regeneration. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4078.
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The embryonic and larval Xenopus peripheral neural retina contains a population of progenitor cells (RPCs), located in the ciliary marginal zone (CMZ). In addition to homeobox transcription factors, such as Rx and Pax6, a number of forkhead transcription factors, including FoxM1, N2, and N4, are expressed in the CMZ. The Xenopus pre-metamorphic retina is capable of regeneration after resection of up to a third of the eye. The purpose of these studies is to investigate the role of forkhead transcription factors in Xenopus RPC development and retinal regeneration.
Morpholino oligonucleotides and synthetic RNAs were injected into 4-celled Xenopus laevis embryos. Embryos were cultured, fixed in paraformaldehyde, and analyzed. For regeneration studies, the nasal-dorsal quarter of the retina was resected from one eye of stage 44 tadpoles. The tadpoles were then fixed after surgery over a period of 30 days. Gene expression was visualized by in situ hybridization using antisense riboprobes and whole fixed embryos or 8 µM sections of paraffin-embedded embryos. Histology was visualized by hemotoxylin & eosin staining of 8 µM sections of paraffin-embedded embryos.
We have confirmed that FoxM1, FoxN2, and FoxN4 are expressed in the CMZ. In particular, FoxM1 is expressed throughout the CMZ, including the most peripheral compartment, containing the most stem cell-like RPCs. FoxN2 and FoxN4 are expressed in other RPCs within the CMZ. Embryos overexpressing FoxN4 exhibited a variety of phenotypes including extra or ectopic retinal tissue and neural tubes, similar to the Rx overexpression phenotype, and/or reduction in eye size. Extra retinal tissue was observed within the optic nerve, optic cup and neural tube. Smaller eyes were primarily observed in embryos injected with higher doses of FoxN4 RNA at early stages. In some cases, FoxN4-injected embryos developed whole ectopic eyes. Ectopic neural tubes contained extra neural progenitor cells. Embryos injected with FoxN4 antisense morpholino oligonucleotide exhibited a reduction in eye size. The RPCs in the CMZ took on an abnormal histological appearance. After partial retinectomy, RPC-like cells appear at the site of regeneration. We have found that these cells express FoxN2 and FoxN4.
This data suggests that FoxN4 is involved in normal development of neural and retinal progenitor cells in Xenopus. Additionally, our data suggests that RPC forkhead transcription factors are expressed in RPCs recruited to the regenerating retina.
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