Figure 1. schlei mutants display multiple developmental defects and reduced primary cilia formation.

e12.5 schlei mutant embryos display broadening of the limbs (A,B) and a subset of mutants also show exencephaly (60%; n=140; bracket in B) or microphthalmia (66% at e12.5 and older; n=29; arrowhead in B). Early limb broadening resolves into preaxial polydactyly (asterisk) as shown in e18.5 hindlimbs (C,D). Confocal Z-stack projections of immunostaining of transverse cryosections through the limb (E,F) and neural tube (G,H) at e11.5 reveals a reduction in cilia number (as marked by Arl13b in green) in schlei animals (F,H) as compared to wild type (E,G) despite the normal appearance and localization of basal bodies (as marked by γ-tubulin in red). (I,J) Scanning electron microscopy confirms a reduction in number of cilia in the neural tube of schlei animals (J) as compared to wild type (I). Cilia that do form in schlei are malformed, including examples with bulges at the tip (red arrowhead), elongated and curled cilia (red arrow), or thin cilia (yellow arrowhead). Differentiated MEFs from e13.5 wild type (K) and schlei (L) embryos immunostained using acetylated α-tubulin reveal a reduction in cilia number in the mutants (cilia, arrowheads). (M) schlei mutant MEF lines showed fewer cilia than wild-type (n=4 lines of each examined). On average, 59.14 ± 6.75% of wild type cells formed cilia, compared to 30.22 ± 4.90% of mutant cells (p=0.00045 by two-tailed student's t-test with equal variances). In (C,D) hindlimbs are visualized with Alizarin red (bone) and Alcian blue (cartilage) staining. Blue staining in (G,H) = phalloidin. Blue staining in (K,L) = DAPI. Control and schlei mutant images are shown at the same magnification.