Fig. 2.

Fig. 2.

Morpholino (MO)-mediated gene knockdown of Ccn2a reduces segment length in regenerating fins. (A) Illustration of unspliced ccn2a mRNA with Ccn2a-MO1 binding site denoted by a red horizontal line. Primer locations shown used to determine whether ccn2a mRNA was targeted. (B) DNA gel showing amplicons using primer set P1 and P2. The slightly larger band (arrow) in Ccn2a-MO injected fins indicates that intron 2 was retained. The last two lanes show DNA amplified using control primers C1 and C2 (n=5 fins pooled together per cDNA, with three biological replicates). (C) Ccn2a-MO1 and SC-MO were injected into one side of the fin and compared to the uninjected side to calculate percent similarity. MO-injected fins were stained using calcein and measured for segment length (double white arrows). Representative images are shown and data are quantified using the percent similarity method (n=24 per treatment group, with two biological replicates). Insets identify individual segments, joints are indicated by white arrows. (D) Graph displays mean±s.e.m. of percent similarity and showed a significant decrease in segment length compared to SC-MO (two tailed, unpaired Student's t-test P<0.0001). (E) In situ hybridization was performed using an antisense digoxygenin-labeled probe against evx1 to measure gene expression. Expression of evx1 is measured by the frequency of positive or negative expression in fin rays denoted by a plus or minus sign. There are more evx1 positive fin rays in Ccn2a-MO1 injected fins (n=5 per treatment, with three biological replicates). (F) Increase of gene expression was quantified through qPCR in both Ccn2a-MO1 and SC-MO. Graph shows a mean±s.e.m. of three biological replicates fold difference (n=5 fins per replicate). A fold difference of 1 means no change from wild-type expression. Student's t-test (two tailed, unpaired) was used to assess significance with a P-value of 0.02. (G) Cartoon illustration of unspliced ccn2a mRNA with Ccn2a-MO2 binding site denoted by a red horizontal line. Primer locations shown used to determine if ccn2a mRNA was targeted. (H) DNA gel showing amplicons using primer set P1 and P2 for MO2 (n=5 fins pooled together per cDNA, with three biological replicates). (I) Ccn2a-MO2 and SC-MO were injected into one side of the fin and compared to the uninjected side to calculate percent similarity. MO-injected fins were stained using calcein and measured for segment length (white arrows). Representative images are shown and data are quantified using the percent similarity method (n=24 for each treatment with two biological replicates). Insets identify individual segments, joints are indicated by white arrows. (J) Graph displays mean±s.e.m. of percent similarity and showed a significant decrease in segment length compared to SC-MO (two tailed, unpaired Student's t-test P=<0.0001). (K) In situ hybridization was performed using an antisense digoxygenin-labeled probe against evx1 to measure gene expression. Expression of evx1 is measured by the frequency of positive or negative expression in fin rays denoted by a plus or minus sign. There are more evx1 positive fin rays in Ccn2a-MO2 injected fins (n=4 per treatment, with three biological replicates). (L) Increase of gene expression was quantified through qPCR in both Ccn2a-MO1 and SC-MO. Graph shows a mean±s.e.m. of three biological replicates fold difference (n=5 fins per replicate). A fold difference of 1 means no change from wild-type expression. Student's t-test (two tailed, unpaired) was used to assess significance with a P-value of 0.02. Scale bar: 100 µm.