DNA sequences driving expression in hair cells are located within Intron 2–3. Transient expression of EGFP placed under the control of a BAC containing various portions of myo7aa gene. Upper panel shows the lateral view of the otic vesicle of a 28 hpf (A,B) and a 44 hpf (C,D) embryo injected with recombinant BAC 29o13-myo7aa_Ex2/SP6:EGFP. A and C are bright field images of the region observed by epifluorescence microscopy in B and D. EGFP is observed in sensory hair cells of the anterior maculae (B,D). Central panel shows the lateral view (E–H) of a 3 dpf embryo and the dorsal view (J–M) of a 5 dpf embryo injected with recombinant BAC 29o13-myo7aa_Ex3/Ex4:EGFP. Transient expression of EGFP was monitored using a Zeiss epifluorescence microscope. E shows the bright field image of the region located directly posterior to the eye; F shows the fluorescent signal of the exact same field observed in panel E. Panel G (superimposition of bright field and fluorescent images) and H (fluorescence only) are high magnification views of the neuromast boxed in panel E and F. (I) model dorsal view of the left ear of a 5 dpf zebrafish on which the different sensory patches are boxed to facilitate identification and recognition of the sites where fluorescent signal is observed in panels J–M. EGFP is observed in sensory hair cells of the posterior maculae (J) and the three cristae (K–M). Lower panel shows the lateral view of a 5 dpf embryo injected with recombinant BAC 29o13-myo7aa_Ex3/SP6:EGFP. (N) model lateral view of the left ear of a 5 dpf zebrafish on which the different sensory patches are boxed to facilitate identification and recognition of the sites where fluorescent signal is observed in panels O–R. EGFP fluorescence is observed in sensory hair cells of the anterior maculae (O), of the anterior and lateral cristae (P,Q) and in a head neuromast (R). Live embryos were anesthetized with tricaine and mounted in agarose before imaging on a Zeiss epifluorescence microscope. All images are single z-axis sections. am: anterior maculae; pm: posterior maculae; ac: anterior cristae; lc: lateral cristae; pc: posterior cristae; n : neuromast.

Expression of EGFP in fish transgenic for B1B2-3Kb and B1B2-761bp DNA fragments. Lateral views of homozygous transgenic zebrafish embryos. Panels A,C,E,G,I,K: line Tg(B1B2-3Kb:EGFP); panels B,D,F,H,J,L: line Tg(B1B2-761bp:EGFP). Each transgenic line was derived from one founder F0 fish: founder #3B for the 3KB DNA fragment (out of 3 F0 founders) and founder #2A for the 761 bp DNA fragment (out of 2 F0 founders). Live embryos were anesthetized with tricaine and mounted in agarose before imaging on a Leica spinning disk microscope. All images are z-axis projections. Embryonic stages imaged were: 30 hpf (A,B), 78 hpf (C–F), 6 dpf (G–L). Each panel comprises the superimposition of bright field with fluorescence image (left part) and the fluorescence image (right part) of the exact same area of the embryo. Panels C–H: arrow = anterior or posterior maculae; arrowhead = anterior, lateral or posterior cristae; * = neuromast. Panels K and L: am = anterior maculae; ac = anterior cristae; lc = lateral cristae; n = neuromast. Scale bar: 20 µm in all panels except for panels C and D (50 µm). C and E as well as D and F represent the same area acquired at a different magnification (20X objective for C and D, 63X objective for E and F).

Expression of EGFP in fish transgenic for B1B2mB-761bp, B1-344bp and B2-431bp DNA fragments. Lateral views of homozygous transgenic zebrafish embryos. Panels A,D,G,J,M,P: line Tg(B1B2mB-761bp:EGFP); panels B,E,H,K,N,Q: line Tg(B1-344bp:EGFP); panels C,F,I,L,O,R: line Tg(B2-431bp:EGFP). Each transgenic line was derived from one founder F0 fish: founder #2A for the B1B2mB-761bp DNA fragment (out of 3 F0 founders), founder #5B for the B1-344bp DNA fragment (out of 3 F0 founders) and founder #7B, the unique F0 founder for B2-431bp DNA fragment. Live embryos were anesthetized with tricaine and mounted in agarose before imaging on a Leica spinning disk microscope. All images are z-axis projections. Embryonic stages imaged were: 30 hpf (A–C), 78 hpf (D–I), 6 dpf (J–R). Panels A,D,G,J,M,P comprise the superimposition of bright field with fluorescence image (left part) and the fluorescence image (right part) of the exact same area of the embryo. For Tg(B1-344bp) and Tg(B2-431bp) transgenic zebrafish, only fluorescent image of the same embryonic area is shown. (D,G,J) arrow = anterior or posterior maculae; arrowhead = anterior, lateral or posterior cristae; * = neuromast. (M) am = anterior maculae; pm = posterior maculae; ac = anterior cristae; lc = lateral cristae. Scale bar: 20 µm in all panels except for panels J (50 µm).

Element B1B2-761bp is a true enhancer and not a cryptic promoter. Transient expression of EGFP placed under the control of B1B2-761bp DNA fragment in native (A,C,E,G,I,K) and inverted (B,D,F,H,J,L) orientation. DNA was injected into wild-type zebrafish embryos at 1-cell stage in absence of Tol2 transposase. Live embryos were anesthetized with tricaine and mounted in agarose before imaging on a Zeiss epifluorescence microscope. All images are single z-axis sections. Embryonic stages imaged were: 30 hpf (A,B), 52 hpf (D–F), 76 hpf (G–L). Scale bar: 50 µm in panels A and B; 20 µm in panels C–L.

Element B1B2-3Kb responds to MIB disruption by an enlargement of EGFP expression area. Expression of EGFP in fish transgenic for the B1B2-3Kb DNA fragment and carrying heterozygous (A,C) or homozygous (B,D) MIB ta52b mutation. Live embryos were anesthetized with tricaine and mounted in agarose before imaging on a Leica spinning disk microscope. All images are z-axis projections. Embryonic stages imaged were: 32 hpf (A,B), 56 hpf (C,D). All panels comprise the superimposition of bright field with fluorescence image (left part) and the fluorescence image (right part) of the exact same area of the embryo. A,C: arrows = anterior and posterior maculae; arrowhead = anterior, lateral and posterior cristae; * = neuromast. Enlargement of EGFP expression domain in MIB ta52b mutants carrying B1B2-3Kb:EGFP transgene correlates with that of myo7aa mRNA. Detection of endogenous myo7aa mRNA in Tg(B1B2-3Kb:EGFP)+/+ MIB ta52b +/ (E-H) and Tg(B1B2-3Kb:EGFP)+/+ MIB ta52b / (I–L) embryos at 24 hpf. (E,I) dorsal view of embryos showing in situ hybridization staining of myo7aa mRNA revealed with NBT/BCIP in the two otic vesicles. (F–H, J–L) double staining for EGFP (immunofluorescence) and myo7aa mRNA (FastRed in situ hybridization) in MIB ta52b +/ (F–H) and MIB ta52b / carrying B1B2-3Kb:EGFP transgene (J–L). Merged fluorescence (F,J), EGFP (G,K) and myo7aa (H,L) visualized in lateral views of otic vesicle. Scale bar: 20 µm (A–L).

Compensation of mariner ty220d -/- mutation by expression of mCherry-Myo7aa fusion protein under the control of B1B2mB-761bp DNA element. Lateral views of homozygous transgenic Tg(B1B2mB-761bp:mCherry-Myo7aa) zebrafish embryos. (A,C,E,G,I) homozygous mar ty220d/ larvae; (B,D,F,H,J) heterozygous mar ty220d+/- larvae. Live embryos were anesthetized with tricaine and mounted in agarose before imaging on a Leica spinning disk microscope. Embryonic stages imaged were: 30 hpf (A,B), 78 hpf (C–F), 6 dpf (G–J). All panels comprise the superimposition of bright field with fluorescence image (left part) and the fluorescence image (right part) of the exact same area of the embryo. (C,D,G,H) arrow = anterior or posterior maculae; arrowhead = anterior, lateral or posterior cristae; * = neuromast. (E,F) am = anterior maculae; ac = anterior cristae; lc = lateral cristae. (A,B) single z-axis section; (C,J) z-axis projection. Scale bar: 20 µm in all panels except for panels B (10 µm).

Compensation of mariner ty220d / mutation by expression of mCherry-Myo7aa fusion protein under the control of B1B2mB-761bp DNA element leads to restoration of sensory hair cell function. (A–D) Recovery of normal hair bundle morphology was observed by staining with Alexa 488-Phalloidin (green) and detection of mCherry-Myo7aa fusion protein expressed in Tg(B1B2mB-761bp:mCherry-Myo7aa) transgene was detected by immunofluorescence (red) in 5 dpf embryos. (A,C) embryos do not express Tg(B1B2mB-761bp:mCherry-Myo7aa) transgene whereas embryos depicted in (B,D) do express the fusion protein. (A and B) embryos carrying endogenous mar ty220d +/+ genotype; (C and D) embryos carrying endogenous mar ty220d -/- genotype. Inlays are high magnification views of a few hair bundles showing the typical shape observed within the imaged lateral cristae. Fixed embryos mounted in agarose were observed on a SP5 Leica confocal microscope. Images are single z-axis sections. Scale bar, 10 µm, applies for the lower magnification of the whole cristae. (E–H) Recovery of FM 1-43 uptake in lateral line hair cells of mar ty220d -/- complemented with mCherry–Myo7aa fusion protein expressed under the control of B1B2mB761bp DNA fragment. (E, G) embryos do not express Tg(B1B2mB-761bp:mCherry-Myo7aa) transgene whereas embryos depicted in (F,H) do express the fusion protein. (E and F) embryos carrying endogenous mar ty220d +/+ genotype; (G and H) embryos carrying endogenous mar ty220d -/- genotype. 5 dpf live embryos were observed on an Olympus Macro Zoom. Scale bar = 250 µm.

Transgene expression is localized to sensory hair cells of the inner ear and lateral line - Morphological assessment.

In order to determine the identity of the cells expressing EGFP reporter gene in the inner ear and lateral line, co-labeling of EGFP (immunohistochemistry) and actin (TRITC-phalloidin) was performed on fixed embryos. Embryos from Tg(B1B2-761bp:EGFP) transgenic line were analyzed at 19hpf (A), 40hpf (B-D) and 60 hpf (E-J). For each developmental stage, EGFP (green) and actin (red) were visualized on an upright spinning disk microscope ; a merge of the two signals is also represented. Inserts show higher magnification of hair cells from the observed sensory patch: tether cells (devoid of hair bundle at 19 hpf) (A), and differentiated hair cells from the anterior maculae (B,F), the posterior maculae (C,G), anterior cristae (E), posterior cristae (H), and neuromasts (D,I,J). Images are z-projections of stacks allowing to visualize together hair cell body and bundle within one sensory patch. Neuromasts are shown entirely (D,I) or only in their most apical part (J). Scale bar = 25 µm.

Transgene expression is localized to sensory hair cells of the inner ear and lateral line - Molecular assessment.

The identity of the cells expressing EGFP reporter gene within in the inner ear and lateral line was also investigated by double immunofluorescence staining of EGFP and HCS-1, a hair cell-specific antigen, performed on fixed embryos. Embryos from Tg(B1B2-3Kb:EGFP) (A-D) and Tg(B1B2-761bp:EGFP) (E-H) transgenic lines as well as wild-type embryos (I-L) were analyzed at 24 hpf (A,E,I), 78 hpf (B-C,FG, J-K) and 6 dpf (D,H,L). For each developmental stage, EGFP (green) and HCS-1 (red) were visualized on an upright spinning disk microscope. Within each panel a merge of the two signals is represented on top, EGFP labeling in the central portion and HCS-1 labeling at the bottom. EGFP and HCS-1 co-localize in differentiated hair cells of the different sensory patches of the inner ear and of neuromasts.

(B,C,F,G,J,K) arrow = anterior or posterior maculae ; arrowhead = anterior, lateral or posterior cristae ; * = neuromast. (D,H,L) am = anterior maculae ; pm = posterior maculae ; ac = anterior cristae ; lc = lateral cristae ; n = neuromast. Scale bar: 20 µm in all panels except for panels B,F,J (50 µm). Images are z-axis projections of z-stacks except for insets on the right hand side of panels D,H,L which are single sections of the lateral cristae. Within each panel, these 3 single sections of the lateral cristae imaged from lateral (upper section) to medial (lower section) are separated 7 µm away from each other.

Acknowledgments
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