Establishment of transgenic lines that report nervous system specific Notch activity based on nort gene regulatory sequence

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Notch signaling is important in development of the vertebrate retina. During zebrafish retinal neurogenesis Notch activity is polarized as demonstrated by the Tg(−3.4 kb her4.3:dRFP)knu2 transgenic line (Del Bene, 2008). Destabilized fluorescent protein (FP) expression driven by the her4 regulatory sequence was elevated in retinal progenitors with apical nuclei. We are interested in determining if her4.3 FP expression is unique to this transgene or general to other Notch target genes. To investigate this phenomenon we generated additional Notch reporter lines using the previously characterized Tp1 promoter, which consists of 12 Notch responsive RBP-Jκ binding sites driving FP (Parsons, 2009). In addition, we established other Notch reporter transgenic lines based on regulatory sequence for the notch-regulated transcript (nort) gene. Expression of nort is augmented by Notch and enriched in neural progenitors, including those of the retina (Tsutsmi and Itoh, 2007). We generated a construct in which FP was expressed from 3.5 kb of genomic sequence directly upstream of nort. Expression of FP from the nort transgene overlapped with endogenous nort expression. Like endogenous nort, we found that FP from the −3.5 kb nort transgene was partially dependent on Notch activity, as demonstrated by morpholino knockdown of Notch receptors or RBP-Jκ. Similar to the her4.3:FP transgene, nort:FP and Tp1bglob:FP were elevated in a subset of retinal cells with apical nuclei. Interestingly, while each transgene showed overlapping expression, distinct temporal patterns were noted. We are currently assessing how each expression pattern relates to cell fate within the retina.