Fig. 5

Knockdown of X. tropicalis Tmem16a. (a) A schematic diagram (not to scale) showing a MO targeted to the splice donor site of exon 2 of X. tropicalis tmem16a pre-mRNA. Primers used to amplify the resulting mRNA fragment via RT-PCR species are labelled f and r. An arrow indicates the position of a premature termination codon (PTC) in intron 2. Start (ATG) and termination (TAG) codons are indicated in exons 1 and 26, respectively. (b) RT-PCR analysis of tmem16a mRNA in embryos injected with MOC and tmem16a splice MO demonstrate a marked reduction of normally-spliced tmem16a mRNA (arrowhead; expected size 381 base pairs in length) in the latter, despite equal loading (amplification of the housekeeping mRNA odc was equivalent in both samples). A larger mRNA species resulting from disrupted splicing is evident in MO-injected tadpoles (asterisk). No other amplicons were detected. Fragment sizes were compared against a standard DNA ladder (left lane; sizes indicated in base pairs). (c) Morphant embryos have mild anterior-posterior (A, P) defects, delayed head development and small heart (H) edemas (arrowhead). (d–e) Injection of tmem16a splice MO caused a complete loss of Tmem16a protein in the plasma membrane of SSCs in the tadpole skin, marked by PNA staining of the vesicles. Orthogonal views (insets) demonstrate this loss at all cellular planes. PNA staining indicates that SSC development is typical in morphants.