Pax2 coordinates epithelial morphogenesis and cell fate in the inner ear

Abstract

Crucial components of the vertebrate eye, ear and nose develop from discrete patches of surface epithelium, called placodes, which fold into spheroids and undergo complex morphogenesis. Little is known about how the changes in cell and tissue shapes are coordinated with the acquisition of cell fates. Here we explore whether these processes are regulated by common transcriptional mechanisms in the developing ear. After specification, inner ear precursors elongate to form the placode, which invaginates and is transformed into the complex structure of the adult ear. We show that the transcription factor Pax2 plays a key role in coordinating otic fate and placode morphogenesis, but appears to regulate each process independently. In the absence of Pax2, otic progenitors not only lose otic marker expression, but also fail to elongate due to the loss of apically localised N-cadherin and N-CAM. In the absence of either N-cadherin or N-CAM otic cells lose apical cell-cell contact and their epithelial shape. While misexpression of Pax2 leads to ectopic activation of both adhesion molecules, it is not sufficient to confer otic identity. These observations suggest that Pax2 controls cell shape independently from cell identity and thus acts as coordinator for these processes.

Fig. 1

Pax2 is required for otic marker expression. Control (A, A′, C, C′, E, E′) or Pax2 MOs (B, B′, D, D′, F, F′, H H′) were electroporated into otic precursors at the 1–2 somite stage. At HH10–12, Eya1 (A–B′, a–b′) and Gata3 (C–D′, c–d′) expression is present in cells carrying control MOs (green in A, a, C, c; arrow heads), but absent in Pax2 MO electroporated cells (green in B, b, D, d; arrow heads). Note in d′: non-invaginated placode on targeted side. Pax2 protein expression is not affected by control MOs (E, E′, e, e′; arrow head), but absent in cells with Pax2 MOs (F, F′, f, f′; arrow head). Note the difference in cell shape of the control and Pax2 MO cells in e (white arrow head) and f (open arrow head). Loss of Pax2 does not affect Sox2 expression in the otic placode (H, H′, h′). Gata3 expression is rescued when otic cells are co-electroporated with splice blocking MOs and a Pax2 expression construct (G, G′, g, g′). Lines in A′–D′, E, F, G′ and H′ indicate the level of sections shown in a–g, a′–g′.

Fig. 2

Pax2 is not sufficient to confer otic identify to ectodermal cells. Pax2-GFP (B, B′, D, D′, green; E, brown) or GFP (A, A′, C, C′, green) was misexpressed at the 0–1 somite stage. While Gata3 expression is induced ectopically (D′, d, d′, arrow head), Eya1 (B′, b′) and Pax2 (E, e′) are not. Pax2 misexpression in the otic placode leads to loss of Eya1 (B′, b; arrow head) and Pax2 (E, e, arrow head), but expression of Gata3 does not change (d). No effect is observed in control electroporated embryos (A′, a, a′, C′, c). Black lines in A′–E indicate the level of sections shown in a–e and a′–e′.

Fig. 3

Cell shape changes during otic placode formation and assembly of the apical junctional complex. A. GPF was electroporated into otic precursors to visualise individual cells. Otic cells elongate dramatically after the 6 somite stage and continue to do so over the next hours. Bottom row: five representative cells from each stage to illustrate elongation. B. The elongation index (length/width ratio; EI) changes significantly between 6 and 7 somites (***p = 0.0007); mean values ± standard deviation are shown. C. N-CAM is absent in otic precursors at 5 somites (open arrow); it is first observed at the 9-somite stage (arrow) and intensifies thereafter. At 16 somites, double staining with phalloidin shows N-CAM (open arrow heads) localisation just basal to apical actin demarcated by the white line in bottom right panel. D. The components of adherens junctions cadherin, α-catenin and β-catenin assemble apically after the 10 somite stage.

Fig. 4

Pax2 controls cell shape in the otic placode. A. Otic precursors were electroporated with control MOs or GFP (top), Pax2 MOs (middle) or Pax2-GFP (bottom). While control cells have elongated at the 13–15 somite stage, Pax2 loss and Pax2 overexpression (Pax2 OE) result in loss of placode cell morphology. Right: five representative cells for each condition. B. Compared to control electroporated cells (WT 13–15ss) the EI is significantly reduced (***) in cells electroporated with Pax2 MOs or Pax2-GFP (Pax2 OE). For comparison measurements from 6 somite placodes are included (WT 6ss). Graph shows mean values ± standard deviation.

Fig. 5

Pax2 is required for N-cadherin and N-CAM expression. A. Control electroporated otic placodes (top row) express N-cadherin (magenta) at the 15–16 somite stage. Loss of Pax2 (Pax2 MO, middle row) leads to loss of N-cadherin (open arrow heads, magenta), while N-cadherin is upregulated (white arrow heads) when Pax2 is misexpressed (Pax2 OE, bottom row). Note: N-cadherin is localised apically. B. Control electroporated otic placodes express N-CAM at the 12–13 somite stage (top row, magenta). Loss of Pax2 (Pax2 MO, middle row) leads to loss of N-CAM (open arrow heads, magenta). In contrast overexpression (Pax2 OE, bottom row) results in increased N-CAM (white arrow heads). Note: N-CAM is localised along the entire cell surface. C. N-CAM is not expressed in trunk ectoderm (control), however ectopic expression of Pax2 (Pax2 OE) in this tissue leads to upregulation of N-CAM in electroporated cells (green, white arrow heads), but not in non-electroporated neighbours (open arrow heads); *indicates somite.

Fig. 6

N-cadherin and N-CAM are required to maintain otic placode morphology. A. Loss of N-cadherin mimics the absence of Pax2. At the 16–18 somite stage, cells carrying control MOs show elongated shape (top row) and N-cadherin expression apically (magenta); panel on the right shows an overview of the same section with both placodes. In contrast, the placode does not thicken or invaginate in N-cadherin knock downs (bottom row, open arrow heads) and cells remain cuboidal. N-cadherin expression is lost (magenta) in the targeted side, but present on the contralateral side (panel on the right). B. Loss of N-CAM mimics the absence of Pax2. Control electroporated cells (top row) are elongated at the 13–15 somite stage and express N-CAM (red) and Pax2 (blue). In contrast, cells carrying N-CAM MOs (bottom row) are round (open arrow head), have lost N-CAM expression (red), but continue to express Pax2 (blue). Panels on the right show a low magnification to include the contralateral placode for comparison. C. Twenty representative cells from control, N-cadherin and N-CAM MO carrying cells show the difference in cell shape. D. The elongation index of cells expressing N-cadherin or N-CAM MOs is significantly reduced when compared to control electroporated cells (WT 13–16ss). For comparison the EI for placode cells from 6 somite embryos is included. Graph shows mean values ± standard deviation.

Fig. 7

Sox2 is not sufficient to rescue cell shape or placode invagination in the absence of Pax2. Otic precursors were electroporated with Pax2 MOs and Sox2 at HH6/7. Cell shape and invagination of the otic placode remain disturbed: compare the non-electroporated control side (left) and the Pax2 MO/Sox2 expressing contralateral side (green, open arrow head or arrow). Pax2 expression (top row, magenta, open arrow head) is absent in electroporated cells; Sox2 does not rescue N-cadherin expression (bottom row, magenta, open arrow). Panels on the right show higher magnification of the targeted area.