1. Piezo-like Gene Regulates Locomotion in Drosophila Larvae
Yufei Hu, Zhilin Wang, Ting Liu, Wei Zhang 
Cell Reports 
Volume 26, Issue 6, Pages e4 (February 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 1369-1377.e4DOI: (10.1016/j.celrep.2019.01.055)
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3. Figure 1 pzl Regulates Drosophila Larval Locomotion
(A) Schematic summary for the pzl gene span and the generation of pzl knockout mutant (pzlKO) via CRISPR/Cas9 system. Top: locations of the four guide RNAs (gRNAs), labeled with different colors. Bottom: the deleted region (red cross) in the gene span.
(B) Confirmation of pzlKO as a null allele by RT-PCR. The transcript expression of pzl in w1118 was used as control (primers are labeled in A with black arrows).
(C) Confirmation of pzlKO as a null allele for transcript expression by qPCR; fly GAPDH was used as the control gene (n = 3).
(D and E) Examples of locomotion trajectories for wild-type (w1118) (D) and pzlKO (E) during 1 min.
(F–H) Characterization of larval locomotion pattern. Average times of head lifting (F), backward waves (G), and turns >90° (H) within 1 min for w1118 and pzlKO (mean ± SEM, n = 15; ∗∗p < 0.01 and ∗∗∗∗p < , unpaired t test).
See also Figure S1 and Videos S1 and S2.
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4. Figure 2 pzl Functions in Cho Neurons
(A) Strategy for generating pzl-Gal4 transgenic flies. Top: pzl gene span and predicted promoter and enhancer regions (highlighted with different colors). Bottom: promoter regions were inserted before the Gal4 coding region, respectively.
(B) pzl-Gal4 labeled Cho neurons in Drosophila larvae body wall (pzl-Gal4; UAS-tdTomato, highlighted with orange squares). Right: lch5 Cho in an abdominal segment.
(C) Relative expression level of pzl in the conditional knockout (Iav-Gal4/+; UAS-Cas9/pzl-gRNA) and rescue (Iav-Gal4/UAS-pzl-GFP; pzlKO) lines through qPCR (GAPDH was used as control, and primers are the same as in Figure 1C; n = 3).
(D–F) Average times of head lifting (D), backward waves (E), and turns >90° (F) within 1 min for pzl-knockdown larvae (iav-Gal4; UAS-pzl-RNAi).
(G–I) Average times of head lifting (G), backward waves (H), and turns >90° (I) within 1 min when pzl was specifically knocked out in Cho neurons.
(J–L) Average times of head lifting (J), backward waves (K), and turns >90° (L) within 1 min when Cho neurons were blocked.
(M–O) Average times of head lifting (M), backward waves (N), and turns >90° (O) within 1 min when pzl was rescued specifically in Cho neurons of pzlKO allele.
(P–R) Average times of head lifting (P), backward waves (Q), and turns >90° (R) within 1 min when human Piezo1 was expressed in Cho neurons of pzlKO allele.
(S–U) Average times of head lifting (S), backward waves (T), and turns >90° (U) within 1 min when mouse Piezo1 was expressed in Cho neurons of pzlKO allele.
Mean ± SEM, n = 15; ∗p < 0.05 and ∗∗∗∗p < (one-way ANOWA with Dunnett’s multiple-comparisons test). n.s., not significant.
See also Figure S3.
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5. Figure 3 Cho Neurons Show pzl-Dependent Response to Displacement
(A) Diagram for mechanically stimulating Cho neurons. In a fillet preparation (outlined with gray hexagon), Cho neurons (black) were exposed for imaging. The cap cells (green) and the connected tendon (aqua) were traced via DIC, and a press (red circle) was delivered to the tendon around the midline (gray dashed line). The diagram only highlights the representative Cho and connected tissue in the abdominal segments (not to scale; a, anterior; p, posterior).
(B) Mechanical stimuli by different levels of body wall displacements (10, 20, and 30 μm) induced Ca2+ level changes in control (blue), pzl-RNAi (red), and pzlKO (gray) larvae.
(C) Representative traces of Ca2+ response of Cho neurons to displacements in control (blue), pzl-RNAi (red), and pzlKO (gray) larvae.
(D) Statistical analysis of the Ca2+ response in Cho neurons of control, pzl-knockdown (Iav-Gal4 > pzl-RNAi), and pzlKO larvae to body wall stimuli (mean ± SEM; ∗p < 0.05 and ∗∗p < 0.01, two-tailed unpaired t tests; n = 6, 6, and 6).
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6. Figure 4 Reconstitution of Pzl Channel Activity
(A and B) Representative traces (A) and summary plots (B) for outside-out patch recordings in response to 50 mm Hg pressure (n = 9 for each transfection; magenta, S2 cells; cyan, HEK293 cells).
(C) Staining of tagged Pzl. Pzl-HA-IRES-GFP was transfected to HEK293 cells (scale bar, 10 μm).
(D) Dorsal-lateral cluster of interneurons in the adult antennal lobe labeled with GH298-Gal4 > UAS-Pzl-GFP. White dashed line outlines the border of the right antennal lobe. Blue probe, recording pipette; orange probe, poke electrode. Red dashed rectangle shows a single focal plane of 0.5 μm (scale bar, 10 μm).
(E) Representative traces of whole-cell recording on antennal lobe LNs (Upper trace: GH298-Gal4>UAS-GFP; Lower trace: GH298-Gal4>UAS-pzl-GFP). Displacement was given at 10 μm for 100 ms.
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