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Fig. 2. Medial versus lateral roof plate cells differ in tissue organization, proliferative capacity and order of emergence from the rhombic lip. (A,B) Doubly transgenic embryos Egr2::cre (r3/r5 cre allele); RC::PFwe (Cre-responsive ßgal indicator allele) X-gal-stained for ßgal activity. Broken lines demarcate the rhombic lip. (A) At E9.5, hindbrain roof plate epithelium (hRPe) cells arising from different rhombomeres intermix anteriorly. (B) At E11.5, medially located (med) hRPe cells are an admixture from different rhombomeres, whereas laterally located (lat) hRPe cells are compartmentalized with respect to rhombomeric origin. (C) Transverse section through an E11.5 doubly transgenic Wnt1::cre; Cre-responsive ßgal indicator embryo so as to distinguish hRPe cells by ßgal activity (green); DAPI is shown in blue. Broken white line highlights the sharp border in tissue organization observed between laterally and medially located hRPe cells. Asterisk indicates a tear in the tissue, resulting in ectoderm cells situating away from mesenchymal cells. Arrowheads indicate laterally located hRPe that is segregated from overlying mesenchyme and epidermal ectoderm. (D-G) Transverse sections through a doubly transgenic Wnt1::cre; Cre-responsive ßgal indicator embryo, with ßgal expression (green by immunodetection) distinguishing rhombic lip and hRPe from mesenchyme and ectoderm; Ki-67 immunoreactivity is in red. Co-labeling was detected in both medially and laterally located hRPe at E9.5 (arrowheads, D,E), but largely absent in medially and laterally located hRPe at E11.5 (F and arrowheads in G). Inset in G shows proliferation (co-labeling) of rhombic lip cells; arrowhead demarcates non-mitotic laterally located hRPe. (H,I) Doubly transgenic Wnt1::FlpeER^T2; R26::FRAP embryos (E11.5) treated for PLAP histochemistry (cells marked by black precipitate). Broken lines demarcate the lower rhombic lip. Induction of recombination by tamoxifen (TAM) administration separates temporal cohorts of Wnt1 (rhombic lip)-derived hRPe. (H) E7.5 TAM administration (E8 recombination in the rhombic lip) resulted in labeled hRPe cells in both medial (med, red arrowheads) and lateral (lat) roof plate locations, albeit a modest number. (I) E9.5 TAM administration (E10 recombination in the rhombic lip) resulted in labeled hRPe cells in only laterally located roof plate. (J) Summary schematic. At E9.5, hRPe cells intermix and are mitotic (yellow). At E11.5, two fields of hRPe are present: (1) cells that are medially located, dispersed and largely non-mitotic that emerged early from the rhombic lip (yellow) and (2) cells that are laterally located, compartmentalized along the anteroposterior (AP) and dorsoventral (DV) axes, are non-mitotic, and that emerged later from the rhombic lip (blue). mb, midbrain; hb, hindbrain; ov, otic vesicle; ect, epidermal ectoderm; mes, mesenchyme; rec, recombination time point; harv, harvest time point; RL, rhombic lip; TAM, tamoxifen.

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