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Figure 3. X. laevis CAV3.2 Morpholino Knockdown
(A) Quantification of MO knockdown phenotype at stages 21 and 27. Two MOs targeting X. laevis CAV3.2 (CAV3.2-MO1 and CAV3.2-MO2) and two control MOs (mismatch [CTL-MO1] and standard control [CTL-MO2]; see Experimental Procedures) were tested. The quantities of MO injected per embryo are indicated below the number (N) of embryos scored for each dose of MO.
(B) Disruption of CAV3.2 transcript splicing by CAV-MO1. RT-PCR was used to detect both the correct and expected splice-disrupted transcripts in cDNA samples from three pooled CTL-MO and three CAV-MO1-injected embryos. Muscle actin (M. Actin) RT-PCR was used to control for RNA load, and RT-minus controls are also shown.
(C and D) Early tail-bud stage embryos (stage 22) injected at the one-cell stage with a CTL-MO1 (C) or a CAV3.2-MO1 (D). Yellow arrowheads in (D) indicate anterior open neural tube.
(E–G) Tail-bud stage (stage 26) CTL-MO1- (E) and CAV3.2-MO1- (F and G) injected embryos. Anterior neural tube defects were observed in the CAV3.2-MO1 injections either with cellular matter erupting from the open neural tube (yellow arrowhead in F) or with a malformed head and deep dimple in the hindbrain (yellow arrowhead in G). See also Movie S2.
(H–J) NCAM staining (red) in anterior or posterior coronal sections of CTL-MO1- (H) and CAV3.2-MO1+2- (I and J) injected embryos.
(K–M) E-cadherin staining (yellow) in anterior and posterior coronal sections of CTL-MO1- (K) or CAV3.2-MO1+2- (L and M) injected embryos. MO-injected embryos used for sections were stage matched at approximately stage 24 based on somite development. All sections were also stained for nuclei (DAPI, blue).
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