Figure 2. Ingression of neurog1⁺ cells.

(A) The otic epithelium and its anterior region at 13 hpf. Arrowheads highlight neurog1⁺ cells outside the otic epithelium. (B) Selected frames of a 3D reconstruction (dorsal view) of the otic placode following the movement of the anterior neurog1⁺ cells. Arrowheads at 14.5 hpf indicate neurog1⁺ cells before epithelialisation (white: cells outside the placode, orange: ingressing cells). At 15.5 hpf red bracket identifies cells that will ingress (shown at 17 hpf) and blue bracket cells that will not ingress. In (A) and (B) the contrast of the red signal was increased to improve visualisation. (C) Selected planes of a 3D tracking of a single cell specifying during ingression (white dot). At 108 min the cell is already epithelialised. Asterisk indicates the SAG. (D–F) 3D tracking of single cells during ingression. (D) 3D reconstruction (dorsal view) showing the initial position of the tracked cells (white, pink and blue dots) at 14 hpf. The violet dot indicates the posterior vertex of the placode. (E) 2D visualisation of the 3D tracks shown in (D) are displayed in a temporal color code. Each track was displaced in the y axis for better visualisation. The track of the posterior vertex of the placode is shown on the right (see also Figure 2D). (F) Selected frames for the cell of the white track. At 150 min the cell is ingressing and completed at 240 min. At 300 min cytokinesis occurs. Membranes are stained with memb-mCherry. Embryos are Tg(actb:H2B-venusFP). (G) Selected planes showing cell-membrane displacements during migration of the cell tracked in (F). White arrowheads indicate protrusion of the cell front and orange arrowheads the position of the nucleus. (H) Schematic representation of the migration and ingression during epithelialisation (see Figure 2—figure supplement 1 for further details). Blue line: laminin, green line: actin layer, red cells: neurog1⁺ cells, red arrows: migration of neurog1⁺ cells towards the otic placode. Scale bars, 20 µm. Dotted lines outline the limits of the otic vesicle.

DOI: http://dx.doi.org/10.7554/eLife.25543.007

Figure 2—figure supplement 1. Morphogenetic features related to ingression.

(A) Whole mount ISH for neurog1 from 13 and 14 hpf Tg(elA:GFP) embryos. This transgenic line expresses GFP in rhombomeres 3 and 5 (asterisks, at early stages rhombomere three express higher levels than rhombomere 5), facilitating the spatial localization of the otic placode. An immunostaining for GFP was performed after the in situ hybridisation. Dotted lines highlight the limits of the hindbrain (red) and the forming otic placode (white). White arrowheads indicate neurog1 expression. (B) Photoconversion of NLS-Eos stained nuclei at 13 hpf in a region anterior to the epithelium in TgBAC(neurog1:DsRedE)n16 embryos expressing memb-GFP. At 20 hpf photoconverted nuclei were observed in neurog1+ cells inside the vesicle (arrowhead). (C) GFP reporting neurod1 expression in the non-ingressing pool of cells at 18 hpf (in the SAG region) from Tg(neurod:GFP) embryos (blue bracket). Embryos are also TgBAC(neurog1:DsRedE)n16 and express memb-GFP. (D) Early stages of otic epithelialisation. Dashed line indicates the epithelialised part of the otic vesicle. Membranes are stained with memb-GFP. (E) Laminin staining at 14 and 22 hpf in transversal and coronal sections. Nuclei are counterstained with DAPI. White arrowheads indicate the forming otic placode. (F) 3D reconstruction (dorsal view) of an otic vesicle and its anterior region at 14 hpf from a Tg(actb1:Lifect-GFP) embryo. The white arrowheads indicate the actin layer that divides latero-medially the tissues lateral to the hindbrain in two regions (white and yellow asterisks, see also reslice 1). Reslices, built from the white bars 1 and 2 shown in the 3D reconstruction, show transversal sections anterior (reslice 1) or at the position (reslice 2) of the otic placode (dashed line). h: hindbrain (dotted line). (G) Cell ingression evaluated using NLS-Eos photoconversion at 13 hpf in neurog1^hi1059 mutant embryos injected at 1 cell stage with memb-GFP and NLS-Eos mRNAs. White arrowheads indicate ingressed cells at 18 hpf. See also Figure 2H for a scheme of the morphological features described in this figure. Scale bars, 20 µm. Dotted lines in (A–C) outline the limits of the otic epithelium/vesicle.

DOI: http://dx.doi.org/10.7554/eLife.25543.008