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First published online 17 September 2008
doi: 10.1242/dev.026674

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Progressive restriction of otic fate: the role of FGF and Wnt in resolving inner ear potential

¹ Laboratory for Sensory Development, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
² Neurogenetics Group, Carl of Ossietzky University, Oldenburg 26111, Germany.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Otic commitment is progressive. (A) The prospective otic region (boxed) was isolated over a number of stages, and cultured in collagen. (B) Histogram showing the number of explants positive for Soho1 and hair-cell antogen (HCA). Greater than 50% of isolates explanted at 7-8ss showed autonomous Soho1 expression and HCA immunoreactivity. Asterisk represents the stage at which the autonomy of differentiation can be considered statistically significant. (C,D) Explants isolated at 5ss do not express Soho1 (C) and do not develop hair cells (D). (E,F) At 7ss, explants express Soho1 (E) and show good hair cell differentiation (arrow, F).

View larger version (57K): [in this window] [in a new window]   Fig. 2. Sustained FGF overexpression stimulates OEPD fate but inhibits otic differentiation. (A) Schematic showing the strategy for electroporation into the anterior primitive streak of HH4 ex ovo cultured chicken embryos. Cathode and anode are shown, dorsal (d) is down and ventral (v) up. (B,C) Unilateral electroporation targets the paraxial mesoderm and the overlying neural ectoderm on one side of the embryo (B). Section shows unilateral electroporation of the mCherry tracer construct revealed by a dsRed antibody (C). (D) Control embryos, electroporated with empty vector show normal Pax2 expression. Line marks the axial level of the section. (E) pEF-Fgf3 extends the Pax2 expression domain (n=10/22). (F)pEF-Fgf19 extends the Pax2 expression domain (n=15/28). (G) Both pEF-Fgf3 and pEF-Fgf19 extend the Pax2 OEPD expression domain (n=15/22). Line marks the axial level of the section. (H) Section taken through the control embryo in D, showing normal Pax2 expression. (I) Section taken through the trigeminal region of the pEF-Fgf embryo in G, showing the unilateral extended Pax2 expression domain. (J) Control embryos, electroporated with empty vector, show normal bilateral Soho1 expression. Line marks the axial level of the section. (K) pEF-Fgf3 reduces the Soho1 domain (n=7/10). (L) pEF-Fgf19 reduces Soho1 expression (n=9/13). (M) Both pEF-Fgf3 and pEF-Fgf19 reduce Soho1 expression (n=8/11). Line marks the axial level of the section. (N) Section taken through the control embryo in J, showing normal bilateral Soho1 expression. (O) Section of the pEF-Fgf electroporated embryo in M, showing unilateral reduction of Soho1 expression. (P) Control embryos, electroporated with empty vector, show normal bilateral Nkx5.1 expression. (Q) pEF-Fgf3 reduces the Nkx5.1 domain (n=1/2). (R)pEF-Fgf19 reduces Nkx5.1 expression (n=5/11). (S) Electroporation of both pEF-Fgf3 and pEF-Fgf19 reduces Nkx5.1 expression (n=5/5). In all cases, the electroporated side is on the right.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Sustained FGF expression increases the number of proliferating OEPD cells. (A) Control embryos showing phosphohistone H3 localisation. The boxed region demarcates the approximate area of the OEPD and cells were counted in this region. (B) Unilaterally pEF-Fgf electroporated embryos transfected on the right side, showing phospho-histone H3 (PHH3) localisation. The number of PHH3-positive cells in the OEPD were counted and compared between the left, unelectroporated,side and the right, electroporated side. (C) Graph showing the difference in the number of PHH3-positive OEPD cells between left and right sides of control and pEF-Fgf electroporated embryos. In control, non-electroporated embryos, the right side of the embryo has 3% fewer PHH3-positive cells (n=565 in 10 embryos) than the left (n=575 in 10 embryos). The right side of experimental embryos, electroporated with pEF-Fgf3 and pEF-Fgf19, has 24% more PHH3-positive cells (n=347 in six embryos) than the left, non-electroporated side (n=261 in six embryos). P-value is <0.001.

View larger version (76K): [in this window] [in a new window]   Fig. 4. Downregulation of Fgf3 and Fgf19 expression downregulates both early and late otic genes. (A) The introduction of shFgf3 downregulates endogenous expression of Fgf3 in the mesoderm of 1-4ss chick embryos. Sections show a reduction of Fgf3 expression in regions that express the mCherry tracer. (B) Introduction of shFgf19 reduces endogenous Fgf19 expression in the mesoderm of 1-4ss chick embryos. Sections show a reduction of Fgf19 expression in regions that express the mCherry tracer. (C) Control embryos electroporated with control shScrambled show no change in Pax2 expression (n=19/21). (D)shFgf3 electroporation results in a slight reduction of Pax2 OEPD expression (n=17/38). (E)shFgf19 causes a slight reduction in Pax2 expression (n=15/30). (F) Electroporation of both shFgf3 and shFgf19 causes a strong reduction of the normal Pax2 OEPD expression domain (n=13/21). (G) Control embryos electroporated with shScrambled show no change in Soho1 expression (only one out of 10 showed aberrant expression). (H)shFgf3 electroporation results in a slight reduction of Soho1 expression (n=8/14). (I)shFgf19 causes a slight reduction in Soho1 expression (n=9/15). (J) Electroporation of both shFgf3 and shFgf19 causes a strong reduction of normal Soho1 otic expression (n=12/19). In all cases, the electroporated side is on the right.

View larger version (107K): [in this window] [in a new window]   Fig. 5. Epibranchial development is modulated by FGF signalling. (A) Chick Foxi2 is normally excluded from the presumptive otic region. (B) Downregulation of Fgf3 and Fgf19 expression following electroporation of shFgf causes a downregulation of Foxi2 expression (n=16/29). (C)pEF-Fgf electroporation results in stronger Foxi2 expression, although it remains excluded from the otic domain (n=11/25). (D) Frozen sections of double in situ hybridisations showing the region of overlap between Pax2 (green) and Foxi2 (red). (E) The region of overlap is reduced or absent when FGF expression is suppressed. (F) FGF overexpression increases the region of Pax2/Foxi2 overlap. (G) At the 20ss of chick development, Pax2 normally segregates into epibranchial and otic expression domains. (H) Downregulation of Fgf3 and Fgf19 results in the reduction of both otic and epibranchial Pax2 domains at 20ss. (I) Sustained FGF expression causes stronger Pax2 expression in both otic and epibranchial domains. (J) By 27ss, epibranchial precursors undergo neurogenesis, forming Phox2b-positive neuroblasts. (K) Epibranchial neurogenesis, as marked by Phox2b expression, is suppressed in shFgf electroporated embryos (n=9/15). (L) In pEF-Fgf electroporated embryos, the Phox2b expression domain is expanded (n=6/9). In all panels the electroporated side is to the right.

View larger version (105K): [in this window] [in a new window]   Fig. 6. Canonical Wnt signal activation inhibits epibranchial formation but does not affect OEPD formation. In all panels, embryos have been electroporated unilaterally on the right-hand side with a constitutively active (CA) β-catenin and a tracer expressing mCherry. (A) Electroporation of CA-β-catenin does not affect the size of the otic placode, as assessed by Soho1 expression. (B) The location of the mCherry tracer in the embryo shown in A indicates widespread electroporation. (C) The expression of Nkx5.1 is not altered in CA-β-catenin-expressing embryos. (D) The location of the mCherry tracer in the embryo shown in C indicates widespread electroporation. (E) Expression of Pax2 in the OEPD is initially normal at 7ss (n=3/4 show no change). (F) Section of the embryo shown in E, showing normal ectodermal Pax2 expression, although some ectopic expression is detected within the neural tube. (G) Section of the embryo shown in E, showing electroporated cells revealed by dsRed immunostaining, which cross-reacts with mCherry. (H) By 13ss, the lateral edge of the Pax2 expression domain is downregulated (n=7/9). Line depicts the level of the section taken. (I) Section of the embryo shown in H, showing reduced lateral ectodermal Pax2 expression. (J) Section of the embryo shown in H, showing electroporated cells revealed by dsRed immunostaining. (K) Foxi2 expression is reduced in response to CA-β-catenin (n=5/6). Line depicts section shown (L,M). (L) Section of the embryo shown in K, showing reduced lateral Foxi2 expression. (M) Section of the embryo in K, showing electroporated cells revealed by dsRed immunoreactivity. (N) Epibranchial neurogenesis, marked by Phox2b expression, is unaffected on the control side of electroporated embryos. (O) Phox2b expression is reduced on the electroporated side of the embryo expressing CA-β-catenin (n=10/12).

View larger version (108K): [in this window] [in a new window]   Fig. 7. Wnt is necessary for inner ear formation before 9ss of development. (A) Soho1 expression is reduced as a result of Dkk1 electroporation (n=12/18). (B) Section shows a reduction of Soho1 and thickened otic ectoderm on one side of the embryo. (C) mCherry tracer expression, as revealed by dsRed immunoreactivity, is unilateral and is seen throughout the ectoderm. (D) Nkx5.1 is also reduced by Dkk1 overexpression. (E) A lateral view of the embryo shown in D, showing widespread mCherry expression throughout the ectoderm. For clarity, the outline of the embryo has been traced. The heart tube (h) can be visualised. (F) Despite unilateral Dkk1 electropoartion, Pax2 expression in the OEPD is initially normal at 7ss (n=8/9 showing normal expression). (G) Sections show normal ectodermal Pax2 expression. (H) Electroporated cells are revealed using dsRed immunostaining. (I) At 13ss, Pax2 is downregulated in a medial portion of the Pax2 expression domain (n=4/5). Line marks the axial level of the section. (J) Section of the embryo shown in I, showing a reduction of Pax2 medially (marked by the bar). (K) Section of the embryo shown in I, showing that electroporation of Dkk1 targets a large extent of the ectoderm, as shown by dsRed immunoreactivity. (L) Foxi2 expression encroaches into the otic territory (n=5/8). (M) Section through embryo shown in L. The region of Foxi2 otic exclusion is reduced by 50%. (N) dsRed immunoreactivity shows the extent of electroporation. (O,P) Epibranchial neurogenesis as marked by Phox2b is not obviously affected as a result of Dkk1 electroporation when control (O) and electroporated (P) sides are compared.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Schematic model of otic and epibranchial development. Competent non-neural ectoderm is acted on by FGF signalling (yellow) to induce Pax2-positive OEPD ectoderm by 5ss of chick development. After 7ss, FGF expression is attenuated and medial Wnt signalling (blue) emanating from the neural tube steers juxtaposed cells to an inner ear fate. Laterally, endodermal FGF signalling together with an unknown signal (red) cause OEPD to be routed into an epibranchial lineage.

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