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First published online 18 July 2007
doi: 10.1242/dev.02874

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Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea

Bonnie E. Jacques^1,2, Mireille E. Montcouquiol^1,*, Erynn M. Layman¹, Mark Lewandoski³ and Matthew W. Kelley^1,

¹ Section on Developmental Neuroscience, Porter Neuroscience Research Center, 35 Convent Dr, Room 2A-100, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA.
² University of Maryland College Park, Department of Biology, College Park, MD, USA.
³ Genetics of Vertebrate Development Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD, USA.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Anatomy of the mouse organ of Corti. (A) Diagram of the lumenal surface of the organ of Corti (OC) at P0. A row of alternating inner hair cells (light green) and phalangeal cells (gray) are bordered by one row of inner PCs (red) and one row of outer pillar cells (PCs, pink). Outer PCs project between first-row outer hair cells (OHCs, olive green). Second and third row OHCs are separated by Deiter's cells (DC, blue). Hensen's cells (HeC, gray) form the lateral edge of the OC. (B,C) Cross-sections of the OC at (B) P0 and (C) adult.

View larger version (124K): [in this window] [in a new window]   Fig. 2. Fgf8 and Fgfr3 are expressed in the developing organ of Corti. In situ hybridizations for Fgf8 and Fgfr3 were performed on E16 (A-D) and P0 (E-G) mice. Fgf8 is not detected at E16 in the apical region (A), but is seen in developing IHCs at the base (B, arrow). (C) At E16, Fgfr3 is faintly detected in a group of cells near the apex (indicated by brackets) and shows more robust expression within a similar region at the base (D). (E) Expression of Fgf8 at P0 is in the IHC (arrow), whereas Fgfr3 (F) is restricted to PCs (arrow) and DCs. (G) Whole-mount of the lumenal surface at P0 illustrating expression of Fgf8 in all IHCs. Scale bars: 30 µm in A-F; 20 µm in G.

View larger version (100K): [in this window] [in a new window]   Fig. 3. Targeted deletion of Fgf8 leads to a disruption in pillar cell development. (A) Lumenal suface of the OC from a littermate control at E18.5. Hair cell stereocilia and cell boundaries labeled with phalloidin (Phal, green) and PCs labeled with anti-p75ntr (red). The row of IHCs and first row of OHCs (OHC1) are indicated. (B) Lumenal surface from an Fgf82,3n/flox; Foxg1cre/+ mouse. Note the disrupted growth of the PCs and close approximation of the IHCs to OHCs. (C,D) Red channels from A and B, respectively, illustrating PC morphology. (D) PCs are missing or underdeveloped in Fgf82,3n/flox; Foxg1cre/+ mice. (E) Cross-section through a control OC at E18.5 showing two PCs (asterisks) extending a lumenal projection between the IHC and first row OHC (numbered). Magnification of the boxed region (inset) illustrates the morphology of the projection, with a red line to indicate the lateral boundary of the IHC and a green line to indicate the medial boundary of the first row OHC. (F) Cross-section through an Fgf82,3n/flox; Foxg1cre/+ OC illustrating a stunted lumenal PC projection (magnified in inset). (G) Average ITO distances (see text for details), as a measure of the degree of PC development, in control and Fgf82,3n/flox; Foxg1cre/+ cochleae. Error bars indicate s.e.m. *, P<0.001. (H) In situ hybridization using a probe specific to the deleted region of Fgf8 in control and Fgf82,3n/flox; Foxg1cre/+ cochleae. Arrowhead points to the row of labeled IHCs in the control; no such labeling is apparent in the Fgf82,3n/flox; Foxg1cre/+ cochlea. Both cochleae have been intentionally over-reacted to ensure complete detection of Fgf8 expression. Scale bars: 20 µm in A-D; 10 µm in E,F.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Inhibition of Fgf8 signaling disrupts pillar cell development. (A) A control cochlear explant that was treated with a function-blocking antibody directed against Fgf5. Stereocilia and cell boundaries are labeled with phalloidin (green) and PCs with anti-p75ntr (red). IHCs and first row OHCs are indicated. (B) Cochlear explant treated with a function-blocking antibody that binds to Fgf8 (labeling as in B). (C) ITO distances for control (anti-Fgf5) and anti-Fgf8-treated explants at basal and midbasal regions of the cochlear duct. *, P<0.001. Scale bar: 20 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Overexpression of Fgf8 induces an increase in p75ntr-positive cells. (A) Low-magnification image of a mouse cochlear explant transfected with a control vector expressing GFP (green). The sensory epithelium (SE) is indicated by expression of p75ntr (red) in the PCs. Kolliker's organ (KO) is located medially to the SE. (B) Similar view as in A, from an explant transfected with an Fgf8 and GFP-expressing vector (green). p75ntr-positive PCs appear normal except in regions located near a cluster of Fgf8-transfected cells (arrows). (C) High-magnification view of a control-transfected cochlea with a large number of transfected cells. (D) High-magnification view of an Fgf8-transfected cochlea. Note the increased number of p75ntr-positive cells. (E) Quantification of effects of expression of GFP or Fgf8 (green pixels) on p75ntr expression (red pixels); see text for details. Control data represent regions with no electroporation in comparison to regions with high GFP or Fgf8 and GFP electroporation. Error bars indicate s.e.m. *, P<0.01. (F) A scatter plot of the relationship between the overall level of transfection (% green pixels) and the overall increase in p75ntr expression (% red pixels). Best-fit linear regression line and correlation coefficient (R2) value are indicated. Scale bars: 100 µm in A,B; 20 µm in C,D.

View larger version (74K): [in this window] [in a new window]   Fig. 6. Fgf17 inhibits development of outer hair cells and Deiters cells. (A,B) Low-magnification view of p75ntr expression (red) in (A) a control explant and (B) in an explant treated with 300 ng/ml Fgf17 for 5 days. (C) High-magnification of a control OC. Cell surfaces and stereocilia are labeled with phalloidin (green), p75ntr-positive PCs in red. (D) High magnification of an Fgf17-treated explant. IHCs are present, but the OHC region appears undifferentiated and is positive for p75ntr (red). (E-F') Lumenal views of the OC from E14 mouse cochlear explants after 6 DIV. (E) PCs, positive for both p75ntr (green) and ß-actin (red), appear as a yellow line between IHCs and OHCs [myosin VI (Myo6) in blue]. (E') ß-actin alone. (F) OHC numbers are reduced and p75ntr expression is increased (green) in the OHC region in Fgf17-treated explants. At the extreme lateral edge, a second band of ß-actin expression (red in F, arrow) appears. (F') ß-actin alone. (G) An Fgf17-treated explant with few OHCs (numbered). Most cells in the OHC region are positive for p75ntr (red). (G') p75ntr expression alone. OHC positions are indicated with numbered circles. (H-I') High magnification of individual examples of OHCs from Fgf17-treated explants. Stereocilia and cell boundaries are indicated in green, p75ntr in red. Asterisks in G-I' indicate cells adjacent to OHCs that have downregulated expression of p75ntr. Scale bars: 200 µm in A,B; 50 µm in C,D; 20 µm in E-F'; 25 µm in G,G'; 20 µm in H-I'.

View larger version (121K): [in this window] [in a new window]   Fig. 7. Fgf17 treatment promotes PC differentiation and induces ectopic PCs. Cross-sections were generated using confocal reconstruction. (A) Cross-section through a control explant showing cell boundaries labeled with phalloidin (green), developing PCs (red) and pillar head (asterisk); OHCs are numbered. HeCs located adjacent to OHC4 are short and have weak p75ntr expression. (B) An Fgf17-treated explant has a broader pillar head (asterisk), reduced OHC numbers, and three rows of PC-like cells in the HeC position (red asterisks). (C,C') High-magnification cross-section of an inner PC (red in C, and shown alone in C') from a control explant. The pillar head narrows as it approaches the lumenal surface. (D,D') Cross-section along the mediolateral plane illustrating the row of inner PCs in a control explant. (E,E') Cross-section along the mediolateral plane illustrating the row of HeCs in the same control explant. Note the shortness of the HeCs and lack of p75ntr expression. (F) Cross-section through an inner PC from an Fgf17-treated explant. Note the increased width of the pillar head. (G,G') Cross-section of PCs from an Fgf17-treated explant, same view as in D. (H,H') Cross section of the HeC region from an Fgf17-treated explant. HeCs are taller, are strongly positive for p75ntr and are morphologically similar to PCs (compare with D and G). (I-L) Examples of PC (I,J) and HeC (K,L) morphology in control (I) and Fgf17-treated (J-L) explants. All illustrate expression of p75ntr. Note the increased thickness of the pillar head in J versus I, and the similarity between endogenous PCs (I,J) and ectopic PCs located in the HeC region (K,L). Asterisks indicate pillar head-like structures. Scale bars: 10 µm.

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