Fig. 1

Pioneer tracking reveals that early OEPD pax2a-positive cells predominantly contribute to the anterior-ventral part of the otic vesicle, including the neurogenic region. (A) Removal of a loxP-flanked transcriptional STOP cassette (red) in the presence of Cre (purple) activates reporter expression (yellow). (B) In a common genetic lineage-tracing setup, Cre expression and reporter activation are directly linked by the constitutive promoter driving reporter expression. Consequently, the reporter is active in all Cre-positive cells even at later stages when Cre expression has vanished. (C) By contrast, use of the conditional temperature-inducible hsp70l promoter uncouples Cre expression and reporter activation. Early activation (red thunderbolt) restricts reporter expression to cells that experience recombination within a nascent Cre domain and allows fate mapping of these cells due to reporter protein persistence at later stages. (D) Late activation (red thunderbolt) of the conditional promoter reveals the fate of the entire Cre domain at later stages similar to a constitutive promoter. (E-G) CreER^T2 (blue) recapitulates the dynamic, endogenous pax2a expression (red) during OEPD stages (3-, 4-, 6-somite) in transgenic Tg(pax2a:CreER^T2)^#31 embryos shown by two-color in situ hybridization. (E2-G2) High magnification views of the OEPD region of embryos shown in E-G, respectively. (H) Schematic of the experimental outline. The progeny of the indicated cross were exposed to tamoxifen (TAM) to elicit immediate Cre-mediated recombination as soon as CreER^T2 is active. Subsequently, the offspring were divided into different groups, exposed to a single heat treatment at various developmental stages [3-, 6-, 9-, 14-somites (so)] and analyzed at 24 hpf. (I,J) EGFP-labeled cells are found in an anterior-ventral position within the otic vesicle after heat shock at the 3- or the 6-somite stage in Tg(pax2a:CreER^T2)^#31; Tg(hsp70l:loxP-DsRed-loxP-EGFP) double transgenic embryos. (K) Heat shock at the 9-somite stage expands the EGFP-positive domain without labeling posterior-dorsal positions within the otic vesicle. (L) EGFP-labeled cells can be detected throughout the otic vesicle after heat shock at placodal stages. E-G are dorsal views with anterior to the top at 3-, 4-, 5- and 12-somite stages. I-L are lateral views with anterior to the left at 24 hpf. Scale bars: in L, 90 μm for E-G; in L, 30 μm for E2-G2 in L, 40 μm for I-L.