Supplemental Figure 1. Analysis of patterning defects in Lef1-bgal embryos.
Analysis of Lef1-bgal expression in heterozygous (b/+) and homozygous (b/b) E9.5 embryos by whole mount staining for lacZ activity. (Panels a,b). Abundant Lef1-bgal expression is observed in the presomitic mesoderm and newly formed somites. LacZ activity is higher in the presomitic mesoderm and is down regulated in the somites, disappearing concomitantly to somite maturation.
Expression of molecular markers by whole mount in situ hybridisation (c-f and k-n), immunohistochemistry (g,h) and morphological analysis (i,j) of wild type (wt) and homozygous Lef1-bgal embryos. (Panels c,d) Pax3 expression in the dermomyotome and the dorsal closure of the neural tube can be detected in the wild type embryo, whereas the spacing of the somitic expression is lost caudal to the forelimb of mutant embryos. Normal Pax3 expression is detected in the dorsal neural tube. (Panels e,f) Wnt3a expression is maintained in the Lef1-bgal mutant embryos in the presomitic mesoderm and the dorsal neural tube as in the wild type embryos. (Panels g,h) Whole mount anti-neurofilament staining of E11.5 embryos show the lack of regularly spaced spinal nerve projections and misrouting of axons in the trunk region and a continuous nerve projection plexus is detected in the caudal part of the embryos. (Panels i,j) Histological analysis of parasaggital sections of E11.5 embryos at the level of the Dorsal Root Ganglia (DRG) shows a regular pattern of condensations in wild type embryos and fusions of the DRGs in the mutant embryos. (Panels k,l) Expression of Semaphorin D (SemD) mRNA by in situ hybridisation is detected in the posterior half of the dermomyotome (white arrowheads), the dorsal neural tube and the limb bud of wild type E9.5 embryos. In mutant embryos, the posterior somatic signal is lost and the pattern of the remaining region shows no discernable SemD signal. (Panels m,n) Whole mount hybridization of E8.5 embryos to detect Dll1 transcripts. Expression of Dll1 is markedly reduced in the presomitic mesoderm of mutant embryos.
Supplemental Figure 2. Electrophoretic mobility shift assay (EMSA) to detect binding of LEF1 to sites in the homology domain-1 and mesoderm-specific enhancer of the Dll1gene. (A) Schematic presentation of the 5’-flanking region of the Dll1 gene, including the homology domains (HD)-1 and -2, the mesoderm enhancer (msd enh) and promoter (msd prom). The positions of LEF1-binding sites are indicated by black boxes. The gray box represents a weak LEF1-binding site. (B) EMSA showing binding of 100 or 300ng purified LEF1 to radiolabeled oligonucleotides, encompassing sites I-II in HD-1. The sequences of wild type (wt) and mutant (mt) oligonucleotides are outlined in Supplemental Table 1A. The binding specificity was confirmed by point mutations of nucleotides in the LEF1-binding sites and by the addition of 100-fold molar excess of unlabelled oligonucleotides encompassing a wild type (wt) or mutated (mt) LEF1 consensus site from the TCRa enhancer. (C) EMSA showing binding of LEF1 to the binding sites III, V, and VII in the msd enhancer of Dll1. Specific binding at levels that are comparable with the binding to the consensus sequence can be detected with all Dll1 oligonucleotides.