DEV01922 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure 1 - Fig. S1. Disruption of the murine mib1 locus and embryonic expression pattern of mib1. (A) The targeting construct is depicted below the endogenous mouse mib1 locus showing five exons (black square). The flanking genomic probe used for Southern hybridization is indicated with a white square. B, BamHI; E, EcoRI; Ev, EcoRV; H, HindIII; DTA, Diphtheria toxin A; IRES, internal ribosome entry site; lacZ, b-galactosidase; puro, PGK-puromycin resistance gene. (B) Southern analysis of EcoRI-digested genomic DNA from targeted ES cell clones probed with the flanking genomic probe shown in A. A 9 kb fragment is detected from the untargeted locus, while the 4 kb fragment indicates the targeted locus. (C) Whole-mount in situ hybridization using a Mib1 antisense probe in an E9.0 wild-type embryo (a) and whole-mount X-gal staining of an E9.0 Mib1^+/- heterozygous embryo (b). Mib1 is expressed broadly throughout the body and abundantly in the branchial arches, presumptive limb buds, and somites. (D) Northern blot analysis during the indicated embryonic stages (E8.5-E15.5) and in ES cells. 28S rRNA was used as a control for sample loading. (E) Western blot analysis of lysates from E9.5 wild-type (wt) and mib1^-/- (mt) embryos. Actin was used as a loading control.

• Supplemental Figure 2 -

  Fig. S2. X-gal staining showing the endogenous expression pattern of Mib1 in E9.5 mouse embryos. (A) The high level of mib1-lacZ expression is evident under the dermomyotome of the somite. (B) In the brain, the expression of mib1-lacZ is observed in the entire neuroepithelium, especially on the pial surface. (C) The endothelium of the dorsal aorta and the surrounding mesenchymal cells show mib1-lacZ positive expression. (D) At the ventral root of the neural tube and the notochord, a mib1-lacZ-positive reaction is observed. (E) The mib1-lacZ expression is shown at the inner surface of the primitive gut. (F) The expression of mib1-lacZ at the mesenchymal cells of the limb bud and the apical ectodermal ridge.

• Supplemental Figure 3 -

  Fig. S3. Whole-mount Dll4 in situ hybridization. (A,B) The aortic expression of Dll4 in wild-type (A; arrows) and Mib1^-/- (B; arrows) embryos.