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. 2013 Oct 23;33(43):17089–17094. doi: 10.1523/JNEUROSCI.0522-13.2013

Figure 2.

Figure 2.

Piezo2b morphants are unresponsive to light touch but respond normally to mechanical and chemical stimuli. Touch response stimulation was performed on 24–27 hpf wild-type and piezo2b morphant embryos. A, wt panel, Time-lapse images of a wild-type embryo's response to touch-evoked stimuli. Piezo2b morphant embryos fail to respond to touch response stimulation (Pz2b MO panel). Trpa1b morphants respond normally to light touch (Trpa1b MO panel). B, RT-PCR analysis of Pz2b-MO3 morphant embryos. The Pz2b-MO3 targets the exon1/intron1 splice site. Primers designed to the start of exon1 and the end of exon 2 (arrows) are able to amplify the correct 270 bp fragment (top band) from wild-type embryo cDNA (lane 1). With morphant embryo cDNA, this band disappears, indicating that the correct splicing of piezo2b has been disrupted (excess primers form the lower band in all lanes). C, Quantitative analysis of wild-type, piezo2b, and trpa1b morphants subjected to touch response stimulation. Piezo2b morphants generated with morpholinos targeting the start codon (Pz2b-MO1), a nonoverlapping morpholino directed toward the 5′ UTR (Pz2b-MO2), or a splice-blocking morpholino targeting the exon1/intron1 boundary (Pz2b-MO3) fail to respond to tactile stimulation. The effect is enhanced slightly by increasing the concentration of morpholinos. Trpa1b morphants (Trpa1b-MO) respond normally in this assay (n = cumulative number of embryos from three independent assays). D, A tail pinch assay was performed on the same wild-type, piezo2b, and trpa1b morphants used in the previous light touch assay. The response of piezo2b and trpa1b morphants was indistinguishable from that of wild-type control embryos (n = cumulative number of embryos from three independent assays). E, Piezo2b morphants respond the same as control embryos when challenged with mustard oil. The assay was performed on the same wild-type and piezo2b morphants used in the previous light touch assay. However, mustard oil fails to elicit a response from trpa1b morphants (Trpa1b-MO; n = cumulative number of embryos from three independent assays). To analyze this phenotype in more detail, time-lapse images were recorded allowing us to count the number of writhings in response to mustard oil stimulation, an example of a wild-type embryo response is shown in F (black arrow indicates when a single writhe has been initiated). G, Piezo2b morphants respond to mustard oil with a similar number of writhings to that observed in wild-type embryos, while trpa1b morphants barely elicit a response to mustard oil (n = 10 embryos analyzed for each condition). Error bars indicate ±SEM. *p < 0.001, two-tailed Student's t test.