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M. Cederlund, P. O’Gaora, B. Sapetto-Rebow, F. Yang, B. Kennedy; A Novel Role for Mab21l2 in Zebrafish Retinogenesis?. Invest. Ophthalmol. Vis. Sci. 2007;48(13):61.
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© ARVO (1962-2015); The Authors (2016-present)
Vertebrate mab21l2 genes are critical for normal eye development. Knockdown of mouse mab21l2 results in a rudimentary retina and knockdown of zebrafish mab21l2 causes a ~ 40% decrease in eye size (Yamada et al, Dev Biol. 2004; Kennedy et al, Dev Biol 2004). In addition to extensive expression in the proliferating vertebrate eye mab21l2 appears to be expressed in restricted layers in the differentiated retina. Our goal is to determine if mab21l2 is required for specific retinal cell fates.
Deletions containing 7.2 kb, 4.9 kb, 3.7 kb or 2.2 kb of the mab21l2 promoter region were cloned upstream of EGFP. Constructs were injected into zebrafish embryos and EGFP expression analysed by fluorescent microscopy. Transgenic lines were made using Tol2 transposition. Transgenic EGFP expression was characterised in whole embryos and retinal cryosections. Putative regulatory elements were identified by bioinformatics using pipemaker, meme and t-coffe. Loss-of-function phenotypes were assayed by injecting mab21l2 antisense morpholinos into transgenic and wild type embryos.
Regulatory domains that control expression of mab21l2 in the tectum, branchial arches, neural tube, fins, heart, forebrain and hindbrain were identified. The pattern of expression for the two largest constructs is very similar to endogenous mab21l2, apart from the unexpected expression in the heart (Kudoh et al, Mech Dev 2001; Wong et al, Mech Dev. 2002). A highly conserved region present in the -7.2 /- 4.9 kb region has no obvious effect on expression. No construct drives transgene expression during the pre-neurogenic phase of eye morphogenesis contrasting with the robust native mab21l2 expression (Kudoh et al Mech Dev. 2001; Wong et al, Mech Dev. 2002). However EGFP expression is detected in the lens around 30 hpf and from 2 dpf fluorescent cells are observed in the inner nuclear layer. mab21l2 morphants exhibit a significant reduction in EGFP positive cells in the inner nuclear layer suggesting a novel role for mab21l2 in retinogenesis.
The deletion constructs drive expression in all expected locations except from pre-neurogenic eye. Preliminary mab21l2 loss-of-function studies show eye, branchial arches, neural tube and heart defects. Future experiments will identify the EGFP positive cells in the inner nuclear layer and their requirement for mab21l2.
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