Pioneer neurog1 expressing cells ingress into the otic epithelium and instruct neuronal specification
- Universitat Pompeu Fabra, Spain
- Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France
Figures
Figure 1
Specification dynamics and morphogenesis of the otic neurogenic domain.
(A,B) Selected frames of a video of an otic placode from a TgBAC(neurog1:DsRedE)n16 embryo shown in (A) 3D reconstructions (dorsal view) and (B) coronal ventral planes. Green in the right schemes …
Figure 2 with 1 supplement
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 …
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 …
Figure 3 with 1 supplement
Local specification and divisions of neurog1 expressing cells.
(A) Selected planes showing DsRedE expression dynamics in locally specified cells (white and blue dots) from TgBAC(neurog1:DsRedE)n16 embryos expressing memb-GFP. Asterisk indicates the SAG. The …
Figure 3—figure supplement 1
Cell division can precede neurog1 expression.
3D tracking of a neurog1− cell (white dot) that divides and subsequently their daughters express DsRedE and delaminate. Dotted lines outline the limits of the otic vesicle. Asterisk indicates the SAG.
Figure 4 with 2 supplements
Ingressing cells instruct local neuronal specification.
(A,B) Laser ablation of neurog1+ cells before ingression. Two different embryos are shown. Images of the otic epithelium and its anterior region at 12.5 hpf just before (A) and after (B) …
Figure 4—figure supplement 1
Calibration and specificity of ablation experiments.
(A) Calibration of cell ablations. A laser pulse (as described in Materials and methods) was applied to embryos expressing H2B-mCherry in a mosaic manner lateral to the neural tube. In example 1, …
Figure 4—figure supplement 2
Late neurogenic phenotypes after ablation and specification analysis of non-proliferative otic placodes.
(A) Z-projection images of the embryos shown in Figure 4, A and B, 8 hr after ablation (21 hpf). The ablated side and their contralateral non-ablated side of the same embryo are shown (images are …
Figure 5 with 1 supplement
FGF control of neuronal specification.
(A–C) neurog1 expression pattern inside the vesicle in embryos incubated in DMSO or SU5402. (A) Images of otic vesicles at 19 hpf incubated from 11 hpf in DMSO or SU5402 (ventral planes). (B) …
Figure 5—figure supplement 1
Analysis of cell division controlled by SU5402 and neurog1 expression in FGF10a mutant embryos.
(A) pH3 immunostainings at 16 hpf in otic vesicles from DMSO and SU5402 treated embryos. Two embryos in each experimental group are shown. The nuclei were counterstained with DAPI. (B) …
Author response image 1
Author response image 2
Number of pH3+ cells at 16 hpf.
* p<0.001, t test. n=12 for neurog1hi1059/neurog1hi1059 and n=11 for neurog1hi1059/+.
Author response image 3
Number of cells in the otic vesicle (upper panel) or the neurogenic region (lower panel) at 18 hpf.
* p<0.05, t test. n=4 for neurog1hi1059/neurog1hi1059 and n=6 for neurog1hi1059/+.
Author response image 4
Cell ingression evaluated using NLS-Eos photoconversion in neurog1hi1059 mutant embryos.
The embryos were injected at 1 cell stage with memb-GFP and NLS-Eos mRNAs. For clarification, the genotype of the embryos was identified after imaging by PCR or phenotyping
Author response image 5
Tracking of photoconverted NLS-Eos nuclei in wild type (A) and hsp70:dnfgfr1-EGFP/+ embryos (B and C).
z-projections of coronal (A and C) or resliced sagittal (B) views are shown. These images were selected from 3D time lapses performed at 5 min time resolution, which allowed us to track individual …
Author response image 6
Cell ingression evaluated using NLS-Eos photoconversion in FGF3 overexpressing embryos.
FGF3 expression was induced with a heat shock by incubating the 11 hpf embryos for 30 min at 39 degrees. For this purpose, the Tg(hsp70:fgf3) line was used. The embryos were injected at 1 cell stage …
Author response image 7
Analysis of tissue folding during placode formation in FGF3 overexpressing embryos.
FGF3 expression was induced with a heat shock by incubating the 11 hpf embryos for 30 min at 39 degrees. For this purpose, the Tg(hsp70:fgf3) line was used. The embryos were injected at 1 cell stage …
Author response image 8
Options 1 and 2 are two different situations that can be underlying the measurements of cell number and cell division.
A and B are the two regions of tissue to be compared. The circles are cells; the green circles indicate mitotic cells. In option 1, the tissue A has much less number of cells and mitosis than tissue …
Videos
Video 1
4D imaging of otic neuronal specification.
3D reconstructed time-lapse of the otic vesicle from a TgBAC(neurog1:DsRedE)n16 embryo. Red: DsRedE fluorescence. Green: memb-GFP. Dorsal view. Time from the first frame is indicated.
Video 2
Specification dynamics visualized in individual cells.
Selected coronal ventral planes from the z-stacks used for 3D reconstructions in Video 1.
Video 3
neurog1 expressing cells locate in the SAG after delamination.
3D reconstruction of the otic vesicle at 21 hpf. White arrow indicates the position of the SAG.
Video 4
Early neurog1 expressing cells located anterior to the otic vesicle. 3D reconstruction of an otic vesicle and the anterior region at 13 hpf, showing the presence of DsRedE expressing cells (white arrows).
https://doi.org/10.7554/eLife.25543.009
Video 5
neurog1 expressing cells ingress in the otic epithelium.
3D reconstructed time-lapse showing the ingression of neurog1 expressing cells. Orange arrowheads indicate ingressing cells and white arrowheads cells that are outside the organ. Cells that will …
Video 6
3D tracking of an individual cell during ingression, division and delamination.
Coronal ventral planes from z-stacks selected to track an ingressing cell (white dot). Note that it begins to express neurog1 before epithelialization.
Video 7
3D tracking of multiple cells during ingression.
Initially, the position of three cells anterior to the otic epithelium is shown (white, pink and blue dots). Tracking (upper panels) and 2D trajectory of each cell (lower panel, yellow track shows …
Video 8
Detailed view of the morphogenesis of the otic placode.
Time-lapse of memb-GFP expressing embryos showing the different stages of tissue epithelialisation. Note that the posterior region folds before the anterior one (orange arrowhead highlights the …
Video 9
Real-time activation of neurog1 expression in local specified cells.
Coronal ventral planes from z-stacks selected to follow the beginning of DsRedE expression in two individual cells that are being specified locally (white and blue dots). Insets show higher …
Video 10
Apical scaffold formation dynamics.
3D reconstructed time-lapse of Pard3-GFP (gray) localization during otic morphogenesis (dorsal view). Pard3-GFP in the otic vesicle (green arrows) or in the superficial external superficial (orange …
Video 11
Coordinated and quick delamination after division of neurog1 expressing cells.
Coordinated delamination: in the upper panel, coronal planes tracking an individual cell before division (white dot) and their daughters after division and until delamination (white and blue dots) …
Video 12
Ablation of pioneer cells before ingression affects neurog1 expression in the neurogenic domain at later stages.
3D reconstruction: DsRedE signal in the neurogenic domain (red) of otic vesicles at 21 hpf corresponding to the previously ablated and contralateral non-ablated sides of the same embryo. A single …
Video 13
Synchronous folding of the anterior and posterior regions of the otic placode in dnfgfr1-EGPF expressing embryos.
Time-lapse during placode morphogenesis in Tg(dnfgfr1-EGFP) embryos heat-shocked at 10 hpf. Lines indicate the epithelial folding.
Video 14
Calibration of cell ablation.
3D reconstruction of z-stacks acquired before and after ablation of embryos expressing H2B-mCherry in some cells adjacent to the neural tube. The neighbouring cells remain undamaged after ablation …
