Fig. 1. Phylogenetic analysis and early expression pattern of XBarhl2. (A) Phylogenetic tree based on identity in 60 amino acid homeobox region between the Barhl proteins in human, mouse, rat, Xenopus, C. elegans and Drosophila and the Barx proteins of human and chicken (Barlow and Francis-West, 1997; Edelman et al., 2000; Hjalt and Murray, 1999). Mouse Barhl2 (Mo et al., 2004), mouse Barhl1 (Bulfone et al., 2000; Li et al., 2002) and XBarhl2 (Patterson et al., 2000; Poggi et al., 2004) sequences were determined by us. Human Barhl2 (GenBank AL355867) and human Barhl1 (GenBank AL354735) sequences were predicted from their genomic DNA sequences; rat Barhl2 (MBH1) (Saba et al., 2003; Saito et al., 1998). The tree was determined using MegAlign from the Lasergene Navigator, licensed by DNAStar. d, Drosophila melanogaster; m, Mus musculus; r, Rattus norvegicus; x, Xenopus laevis; d-rer, Dario rerio; ce, Caenorhabditis elegans; h, Homo sapiens. (B) Expression of Xbarhl2 as determined by RT-PCR analysis on embryos at different embryonic stages and dissected parts of embryos. (a) Xbarhl2 starts to be expressed at stage 10.5. XEF-1α was analyzed as a control. (b) At stage 10.5 Xbarhl2 is expressed in the dorsal part of the embryo. (C,D) Whole-mount in situ hybridization of embryos, using Xbarhl2 as a probe. (C) Dorsal view of stage 14 embryos showing expression of Xbarhl2 in the dorsal diencephalon. (D) Same embryo as C rotated showing expression along the midline (arrow). A, anterior; P, posterior. (E) RT-PCR analysis. (a) Drawing of a stage 14 embryo showing dissected parts (Nieuwkoop and Faber, 1967). (b) Xbarhl2 is expressed in the posterior neural plate, posterior dorsal mesoderm and in the midline of stage 14 embryo. Chordin and Ncam were analyzed as mesodermal and neuroectodermal markers, respectively.
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