The sensory primordium in the cochlea is marked by p27^Kip1 and the zone of non-proliferating cells (ZNPC). Timed-mated pregnant animals were injected with BrdU at E12.5 (A,B), E13.5 (C,D) and E14.5 (E,F), and sacrificed 8 hours later. Adjacent sections from each stage were stained with antibody to either BrdU (A,C,E; green) or p27^Kip1 (B,D,F; red). (A,B) At E12.5, BrdU stained nuclei are seen throughout the cochlear epithelium, including at the site of future organ of Corti formation (A). p27^Kip1 is not expressed at this time (B). The site of future organ of Corti formation (arrows) was determined by staining for spiral ganglion cell axons containing βIII-tubulin or neurofilament (data not shown). (C,D) At E13.5, BrdU-stained nuclei are absent from a region of the cochlear epithelium (brackets), here termed the zone of non-proliferating cells (ZNPC) (C). In an adjacent section, the ZNPC can be seen to correspond precisely with the expression domain of p27^Kip1 (D). (E,F) At E14.5, the ZNPC is still present (E) and the p27^Kip1 staining persists (F). Scale bar: 50 μm.

The onset of Math1 expression in the cochlea. (A-C) Sections from E12.5 (A) and E13.5 (B) were stained with antibody to Math1 (Ab-Math1). No Math1 expression was visible in the cochlear epithelium (outlined in white) before E13.5, although cells in the vestibular system were stained by E12.5 (A,B). A section through the E13.5 cochlea, parallel to the cochlear duct was double labeled with antibodies against BrdU (green) and EGFP (red) (C). The apical to basal extent of the ZNPC is revealed by the BrdU-negative staining region (brackets) where no Math1/EGFP expression is detected, indicating the ZNPC is established prior to the onset of Math1/EGFP expression. (D-F) Sections through E14.5 cochlear epithelium. Sections were labeled with antibody to Math1 (D, red), double-labeled with antibodies to EGFP (E, red) and p27^Kip1 (E, green), or double-labeled with antibodies to EGFP (F, red) and BrdU (F, green). Math1 staining first appears on E14.5 as a column of cells spanning the cochlear epithelium (D, arrow). Math1/EGFP cells (arrows in E,F) appear at the medial border of the p27^Kip1-stained domain (E, bracket) or the ZNPC (F, bracket). Scale bars: in A, 50 μm in A-C; in D, 50 μm in D-F.

Math1expression is restricted to postmitotic cells in the cochlea. Timed-mated pregnant Math1/EGFP transgenic animals were injected with BrdU at either E12.5 (A) or E13.5 (B) and then sacrificed at E14.5. Sections through the cochlear epithelium were double labeled with antibodies against BrdU (green) and EGFP (red). Cells double labeled with both antibodies are yellow. Brackets indicate the sensory primordium. (A) Section through an E14.5 cochlea exposed to BrdU on E12.5. Math1/EGFP cells (arrowheads) are double labeled with BrdU (yellow) indicating that on E12.5, cells that are destined to become Math1/EGFP positive are still in the cell cycle and incorporating BrdU. (B) Section through an E14.5 cochlea exposed to BrdU at E13.5. No double-labeled cells are present, indicating that at E13.5, cells that are destined to become Math1-positive (red, indicated by arrowheads) at E14.5 have exited the cell cycle and do not incorporate BrdU. Scale bar: 50 μm.

A gradient of Math1 expression precedes the appearance of the early hair cell differentiation marker, MyosinVIIa. (A-D) Sections through the developing cochlear duct stained with antibody to Math1. (E,F) Sections stained with antibody to MyosinVIIa. (G,H) Sections double-labeled with antibodies to MyosinVIIa (red) and p27^Kip1 (green). (A,B) At E14.5, Math1 expression has spread to the medial region of the cochlear duct (A) from its site of initiation in the base (B, see Fig. 1 for reference). In the medial region (A), a single column of cells spanning the cochlear epithelium is seen, while in the base (B) two columns of Math1 stained cells are observed (arrowhead, medial; arrow, lateral). The Math1⁺ columns are separated by a row(s) of Math1^– cells. (C-H) One day later, at E15.5, the gradient of Math1 expression has progressed and two columns of Math1⁺ cells are seen in the apical region of the cochlear duct (C, arrowhead, medial; arrow, lateral), and Math1⁺ cells in the mid-basal region (D) have begun to resolve into the stereotyped pattern of one row of inner (arrowhead) and three rows of outer (bracket) hair cells. In contrast to the Math1 staining observed in the base (C,D), no MyosinVIIa staining is observed in adjacent sections in the apex (E), while only a single inner hair cell is stained with MyosinVIIa in the base (F, arrowhead), indicating that Math1 expression precedes that of MyosinVIIa. Sections double-labeled with antibody to p27^Kip1 (green) and MyosinVIIa (red) reveal the change from the thickened epithelium that is present in the undifferentiated apical region (G) compared with the bilayered epithelium in the differentiated organ of Corti (H). Scale bar: 50 μm.

The ZNPC forms normally in wild-type and Math1^–/– embryos. Adjacent sections from E14.5 embryos were stained with antibody to BrdU (A,B), p27^Kip1 (C,D) or Jagged1 (E,F) to reveal the formation of the zone of non-proliferating cells within the cochlear duct of wild-type (A,C,E) and Math1^–/– (B,D,F) animals. BrdU was injected into timed-mated pregnant females 8 hours prior to sacrifice. Brackets indicate the ZNPC seen as the absence of BrdU staining in cells that have withdrawn from the cell cycle (A,B), and the site of p27^Kip1 staining (C,D). Brackets in E,F were placed after overlaying sections A-D above. The Jagged1-positive domain is largely medial (left) to the ZNPC, but appears to overlap the ZNPC slightly at its lateral edge. Arrows in E,F indicate the boundaries of the Jagged1 expression domain. Scale bar: 50 μm.

A gradient of apoptosis in Math1^–/– organ of Corti parallels the normal base-to-apex gradient of hair cell differentiation. Sections were prepared from wild type [A-F, (WT)] and Math1^–/– [G-L (KO)] littermates at E15.5 (A-C,G-I) and E17.5 (D-F,J-L). Apical regions (A,D,G,J) of the developing organ of Corti were compared with basal regions (B,C,E,F,H,I,K,L) in order to observe the gradient of differentiation. All sections were stained with antibody to p27^Kip1 to reveal the sensory epithelium (green). In addition, sections were double-labeled with antibody to either Activated Caspase 3 (ActCasp3) (red; A,B,D,E,G,H,J,K) to reveal apoptotic cells, or MyosinVIIa to reveal the state of hair cell differentiation (red; C,F,I,L). Brackets indicate the extent of the p27^Kip1-containing region, arrowheads indicate inner hair cells and arrows indicate outer hair cells. Scale bar: 50 μm.