FIGURE SUMMARY
Title

Characterization of new otic enhancers of the pou3f4 gene reveal distinct signaling pathway regulation and spatio-temporal patterns

Authors
Robert-Moreno, A., Naranjo, S., de la Calle-Mustienes, E., Gomez-Skarmeta, J.L., and Alsina, B.
Source
Full text @ PLoS One

Temporal expression pattern of GFP driven by pou3f4 HCNR 81675 and HCRN 82478 enhancers.

(A) Schematic representation of the POU3F4 locus in the human chromosome X (hg18 alignment) showing the position of the different inner ear enhancers relative to the POU3F4 coding sequence. (B–C) Onset of GFP protein expression in HCNR 81675 (B) and HCNR 82478 (C) transgenic embryos. Expression in the otic territory occurs at 13.5 hpf in HCNR 81675 and at 18.5 hpf in HCNR 82478 zebrafish embryos (white arrows), GFP in mesonephros and midbrain-hindbrain boundary (red arrows) is also detected in HCNR 82478 embryos. (D–E) Dorsal views of HCNR 81675 (D) and HCNR 82478 (E) transgenic embryos assayed by in-situ hybridization for GFP mRNA expression. In both cases GFP mRNA was detected in the otic field 2 hours before GFP protein was found (B–C). Orientation of the embryos is anterior (left) to posterior (right).

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Terms:
Stage Range: 1-4 somites to Prim-5

Spatial-temporal expression pattern of pou3f4 enhancers in the inner ear.

(A–F) Lateral views of inner ears from zebrafish transgenic embryos for HCNR 81675 (A–C) and HCNR 82478 (D–F) enhancers analysed from 24 hpf to 72 hpf. In HCNR 81675 embryos at 24 hpf, GFP is observed in two broad domains comprising the sensory territories as observed by the otolith deposition (stars) (B). In HCNR 82478, GFP is already restricted to the anterior and posterior sensory macula from its onset as observed by GFP fluorescence relative to the otolith position (star). (C and F) GFP is found in the three sensory crista in 3-day old embryos in both transgenic zebrafish lines. Orientation is anterior (left) and dorsal (up). (G and H) Confocal transverse images of inner ear sensory patches immunostained with the anti-Pax2 antibody in 72 hpf embryos. In HCNR 81675 embryos, GFP is found in supporting cells but absent in hair-cells (Pax2 positive cells; pointed by a white arrow) (G). In contrast, HCNR 82478 embryos displayed GFP in supporting cells but also in hair-cells at lower levels (white arrow) whereas other hair-cells where completely devoid of GFP expression (red arrow). (I and J) Transverse confocal images of sensory patches of both enhancer embryos immunostained for GFP after the injection of the hair cell specific labelling marker FM 4-64FX. The same result was obtained in this experiment. (I) GFP is devoid in FM 4-64FX stained hair-cells in HCNR 81675 embryos (white arrow), whereas some hair-cells displayed GFP in HCNR 82478 embryos (J; white arrow). (K and L) Confocal images taken from the transverse section anterior to the first section from the otic vesicle. Co-immunostaining for anti-GFP and anti-islet1 protein reveals that only in HCNR 82478 transgenic embryos GFP is activated in the otic ganglion (L). (G–L) Lateral (left) and dorsal (up).

Distinct signaling pathways regulate activation of pou3f4 HCNR 81675 and HCNR 82478 enhancers.

(A–N) Transgenic embryos for both enhancers were treated with different pharmacological inhibitors from 5.5 hpf stage to 18–20 hpf and 7.5 hpf to 36–40 hpf respectively. Lateral view of HCNR 81675 (A–G) 18–20 hpf staged otic vesicles and HCNR 82478 (F–N) otic vesicles of 36–40 hpf embryos. HCNR 81675 activity was abrogated in the presence of RA signaling inhibitor DEAB (compare D to the control treatment with DMSO in A), whereas Fgf signaling inhibition by SU5402 completely disrupted pou3f4 HCNR 82478 activity (compare I to control treatment in H). Orientation is anterior (left) to posterior (right). (O, P) Graphs representing the percentage of embryos displaying complete inhibition of GFP expression in pou3f4 HCNR 81675 (O) and HCNR 82478 (P) transgenic embryos after specific signaling pathway blockade. The total number of embryos counted in three independent experiments is represented.

The POU3F4 HCNR 81728 enhancer is regulated by Hedgehog signaling.

(A and B) Lateral view of GFP otic expression in 96 hpf embryos transgenic for the HCNR 81728 enhancer in control (A) and Cyclopamine A treated embryos (B). (C) Percentage of GFP expressing area in otic vesicles from 95% EtOH and Cyclopamine A treated embryos.

EXPRESSION / LABELING:
Gene:
Fish:
Condition:
Anatomical Term:
Stage: Day 4

Co-localization of Pax2 and Sox2 with GFP driven by the HCNR 81675 enhancer.

(A–D) Dorsal view of transgenic embryos assayed by ISH for the expression of pax2a (A), sox2 (B), sox10 (C) at 13, 15 and 18 hpf. GFP (D) displays a similar pattern than sox2 and pax2a at 15 hpf (compare A and B with D). Orientation is anterior to the left. (E–E") Double immunostaining with anti-Pax2 (E) and anti-GFP antibody (E′) in transverse sections of 15 hpf otic vesicles revealed co-localization of both proteins (E"). (F–F") GFP protein (F′) also co-localizes with sox2 mRNA (F") but not sox10 or tbx1 mRNA (G" and H"). (I–P) pax2a and sox2 expression is abolished in retinoic acid treated HCNR 81675 embryos (compare J and L to I and K, respectively) but not other genes such as sox10 or neuroD (compare N and P to M and O, respectively). Dorsal view, orientation is anterior to the left.

Pax2 and Sox2 proteins are required for HCNR 81675 enhancer activation.

(A) Scheme showing wild-type Sox and Pax2/5/8 consensus in the pou3f4 HCNR 81675 sequence and above each one, the mutation in the primers designed for site-directed mutagenesis of the Sox and Pax2/5/8 binding sites. (B–C) Transgenic embryos carrying GFP under the control of the HCNR 81675 enhancer. (B) GFP expression promoted by the wild type HCNR 81675 sequence. (C) GFP expression promoted by the HCNR 81675 enhancer harbouring the double mutation for Pax2/5/8 and Sox binding sites.

HCNR 81675 activity is not dependent on RA, Fgf, Notch, Bmp and Hh at 7.5 and 9.5 hpf. (A–N) GFP is observed after treatment of HCRN 81675 transgenic embryos with pharmacological inhibitors of signaling pathways at 7.5 hpf (A–G) and 9.5 hpf (H–N). Orientation is anterior to the left and dorsal up.

Abrogation of different signaling target genes after treatment with specific signaling inhibitors. (A–J) In situ hybridization for the Fgf, Notch, Retinoic Acid, BMP and Sonic Hedgehog target genes pea3 (A–B), neuroD (C–D), krox20 (E–F), msxC (G–H) and ptc1 (I–J) to confirm inhibitor activity at our working concentrations. Dorsal view, orientation is anterior to the left.

HCNR 82478 activity is dependent on Fgf signaling when treated at 5.5 and 7.5 hpf. (A–N) GFP is inhibited after treatment of HCRN 82478 transgenic embryos with 30 μM SU5402 at 5.5 hpf (A–G) and 50 μM SU5402 at 7.5 hpf (H–N). Orientation is anterior to the left and dorsal up in all images.

Endogenous expression pattern of pou3f4/Pou3f4 in Xenopus and mouse. (A–B) In situ hybridization for pou3f4 mRNA in Xenopus embryos of stage 35 (A) and stage 42 (B). Note that the endogenous expression is detected at the periotic mesenchyme at stage 42, whereas at stage 35 pou3f4 is still not expressed. (C–D′′) In situ hybridization for Pou3f4 mouse mRNA in mice embryos of stage E8.5 (C) and E16.5 (D). In mice, also Pou3f4 is expressed at the otic mesenchyme at later stages, shown in insets (D′, D′′). Transverse sections shown in all panels.

Acknowledgments
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