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Fig. 3. Misexpression of xCcdc11 impairs L–R patterning. To study the role of CCDC11 in
frogs, we knocked down its expression using xCcdc11–MO. Embryos were injected at the 2- to
4-cell stage and analyzed for xCoco expression by whole-mount in-situ hybridization on St. 20
GRPs. Embryos injected with control–MO or uninjected embryos served as controls. In normal
development, xCoco expression on the left is inhibited (blue arrowhead in A), while it continues
to be expressed on the right (A). When leftward flow is defective, xCoco expression may be either
uniform, reflecting no inhibition at all (B), or reduced on the right side (blue arrowhead in
C). Analysis of embryos injected with xCcdc11–MO revealed disruption in the proper inhibition of
xCoco expression. While only 25% of the embryos injected with control–MO exhibited deviation from
normal expression, 49% of xCcdc11–MO-injected embryos showed abnormal expression (P < 0.05). To verify that the laterality disorders induced by xCcdc11 knockdown persist with time, we manipulated xCcdc11 levels and evaluated L–R patterning by in-situ hybridization staining for xPitx2 during the tailbud stages (blue arrowheads in E–H). xPitx2 is normally expressed unilaterally on the left, thus it serves as a reliable marker for laterality (E). Laterality defects may result in either bilateral (F), completely absent (G) or right expression (H) of xPitx2. Analysis of xCcdc11 downregulation by xCcdc11–MO injections resulted in laterality defects, as exhibited by abnormal expression of xPitx2 ( I, P < 0.001). The ambiguous phenotype obtained in xCcdc11 knockdown mimics the diverse phenotype between the two siblings carrying the mutation, and is often observed when GRP flow is disrupted. Overexpression of xCcdc11 also resulted in abnormal expression of xPitx2 (J, P < 0.01), indicating the need for tight regulation of CCDC11 levels for proper L–R patterning. The efficiency xCcdc11–MO in blocking translation of xCcdc11 mRNA was assessed by western blot analysis of total proteins extracted from embryos injected with xCcdc11–MO, uninjected embryos, and embryos injected with control–MO. Analysis with anti-CCDC11 antibody revealed that xCcdc11–MO inhibits xCcdc11 expression by 84% compared to control–MO-injected embryos. Ponceau S total protein staining of the same membrane was used to validate equal loading in all lanes (K). |
