Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification

Abstract

Xenopus is ideal for systematic decoding of cis-regulatory networks because its evolutionary position among vertebrates allows one to combine comparative genomics with efficient transgenic technology in one system. Here, we have identified and analyzed the major enhancer of FoxE3 (Lens1), a gene essential for lens formation that is activated in the presumptive lens ectoderm (PLE) when commitment to the lens fate occurs. Deletion and mutation analyses of the enhancer based on comparison of Xenopus and mammalian sequences and in vitro and in vivo binding assays identified two essential transcriptional regulators: Otx2,a homeodomain protein expressed broadly in head ectoderm including the PLE,and Su(H), a nuclear signal transducer of Notch signaling. A Notch ligand,Delta2, is expressed in the optic vesicle adjacent to the PLE, and inhibition of its activity led to loss, or severe reduction, of FoxE3 expression followed by failure of placode formation. Ectopic activation of Notch signaling induced FoxE3 expression within head ectoderm expressing Otx2, and additional misexpression of Otx2 in trunk ectoderm extended the Notch-induced FoxE3 expression posteriorly. These data provide the first direct evidence of the involvement of Notch signaling in lens induction. The obligate integration of inputs of a field-selector (Otx2)and localized signaling (Notch) within target cis-regulatory elements might be a general mechanism of organ-field specification in vertebrates (as it is in Drosophila). This concept is also consistent with classical embryological studies of many organ systems involving a `multiple-step induction'.

Evolution of a tissue-specific silencer underlies divergence in the expression of pax2 and pax8 paralogues

“…The Xenopus laevis system was chosen to examine the potential enhancer activity of the CNSs, because an efficient transgenesis technique for a non-mosaic founder assay is readily available 17 . Each X. tropicalis pax8-CNS was cloned into a green fluorescent protein (GFP) reporter plasmid carrying a β-actin basal promoter 18 . Each construct was then used to generate transgenic embryos, and their GFP expression was examined once the resulting embryos reached the tailbud stages (stages 30-32) 19 .…”

“…The reporter plasmid, actGFP, carrying a chicken β-actin basal promoter ( − 55 to + 53), was previously described as βGFP 18 . The construct, gloGFP, was generated by replacing the β-actin basal promoter of actGFP with a human β-globin basal promoter ( − 37 to + 12) 20 .…”

“…JQ902140). The reporter constructs carrying the pax2, pax8 or pax2/5/8 promoters were generated by introducing each fragment into the promoter-less GFP reporter plasmid, pBSSK + EGFP 18 . For generating the pax8-GFP fosmid construct, a GFP-flpe-kan cassette was introduced in-frame into a X. tropicalis fosmid clone, AOPZ250282 (European Xenopus Resource Centre), using homologous recombination technique 39 .…”

“…Transgenic Xenopus embryos were generated by a sperm nuclear transplantation method with oocyte extracts 17,41 . The manipulated embryos were cultured until the tailbud stages, and all normally developed embryos were subjected to in situ hybridization to examine their GFP expression with maximum sensitivity 18 . The frequency of GFP expression varied depending on constructs, egg quality and preparation of the oocyte extracts.…”

Xhairy2 functions in Xenopus lens development by regulating p27xic1 expression

“…Only the embryos showing X-gal-positive cells in the head ectoderm including LP were subjected to whole-mount in situ hybridization (WISH). Two important transcriptional regulator genes for lens development, foxE3 (or Lens1 in Xenopus; Kenyon et al, 1999;Ogino et al, 2008) and L-maf (Ogino and Yasuda, 1998;Ishibashi and Yasuda, 2001), are expressed in LP. Co MO injection did not have any significant effects on foxE3 (no change in 93%, n ϭ 27; Fig.…”