Fig 1.
Notch signaling is maintained in prospective neurons.
(A) Schematic representation of the Hes5-VNP sequence that was inserted in the Notch reporter transgenic chick line. (B) Left: Transverse sections of the NT of the Hes5-VNP transgenic line at E3 and E4 immunostained for Venus (green) and HuCD (red) to label neurons. Middle: Color coded map of Hes5-VNP intensity. The red line separates HuCD− from HuCD+ cells. The black dotted lines delineate the ventral limit of the roof plate and dorsal limit of the motor neuron domain. Right: Distribution of the Hes5-VNP signal intensity in HuCD− and HuCD+ cells. Note that cells within the limits of the black dotted lines of the color code panel were labeled in black in the HuCD− population. (C) Top: Time course of the protocol. Bottom: Distribution of the Hes5-VNP signal intensity in FT+/HuCD− cells. This population is then divided into EdU+ (blue) and EdU− (magenta) cells. A minimum of 58 cells collected from four embryos were analyzed for each group. (D) Left: Transverse sections of the dorsal NT in the Hes5-VNP transgenic line at E4 immunostained for Venus (green), Neurog2 (red), and HuCD (blue). Bottom: Enlarged view of the boxed area showing representative examples of Neurog2+ cells. Right: Distribution of the Hes5-VNP signal intensity in Neurog2− and Neurog2+ cells. The latter population was divided based on Neurog2+ signal intensity. A minimum of 75 cells collected from six embryos were analyzed for each group. Horizontal bars correspond to medians. ns, p > 0.05; **p < 0.01, ***p < 0.001 (Kruskal-Wallis test). Underlying data are provided in S1 Data. Scale bar represents 25 μm. See also S1 and S2 Figs. E, embryonic day; EdU, 5-ethynyl-2′-deoxyuridine; FT, FlashTag; Hes5, Hairy and Enhancer of Split 5; HuCD, neuron-specific RNA-binding proteins HuC and HuD; Neurog2, Neurogenin 2; ns, nonsignificant; NT, neural tube; VNP, Venus-NLS-PEST.
Fig 2.
Sequence of events leading to neuron delamination.
(A) Left: Apical view of the NT electroporated at E2 with ZO1-GFP/iRFP (green), along with the constructs indicated on the left, and harvested at different hae, followed by an immunostaining for N-Cadherin. The boxed area indicates the cell of interest. Right: Quantification of the apical area ratio (ratio of the area of one transfected cell versus the mean area of four of its close non-transfected neighbors) and N-Cadherin level ratio (ratio of the average pixel intensity within the apical circumference of one transfected cell corrected by the background versus the mean of average pixel intensity of four of its close non-transfected neighbors) at different hae. Data represent mean + SEM. (B) N-Cadherin intensity ratio as a function of apical area ratio at 24 hae. Data were taken from (A). The “median” used as a threshold to discriminate between small and large apical areas corresponds to the median of the control (0.62). **p < 0.01; ***p < 0.001 (one-way ANOVA). (C) Top: Apical view of the NT transfected with ZO1-iRFP (green) along with the indicated constructs and immunostained for Tuj1 (red) and Par3 (blue). Bottom: Three-dimensional view of the cell represented above but showing only the ZO1-iRFP and Tuj1 stainings. Right: Scatterplot of the mean apical area ratio for Tuj1+ cells. Each point represents one apical area ratio calculated as in (A). n = 49, 66, 51 cells collected from five embryos were analyzed for control, Neurog2, and ΔMaml1, respectively. ns, p > 0.05; ***p < 0.001 (Kruskal-Wallis test). Horizontal bars correspond to means. Underlying data are provided in S1 Data. Scale bar represents 2 μm. See also S3 Fig. ΔMaml1, dominant-negative Mastermind-like 1; E, embryonic day; EP, electroporation; GFP, green fluorescent protein; hae, hour after electroporation; iRFP, infrared fluorescent protein; Neurog2, Neurogenin 2; ns, nonsignificant; NT, neural tube; Par3, Partition defective protein 3; ZO1, Zonula Occludens 1.
Fig 3.
Effects of Notch signaling and apical constriction modulators on apical markers and tissue integrity.
(A) Transverse sections of the NT transfected at E2 with the indicated constructs, harvested at E3 and immunostained for N-Cadherin (red). (B) Transverse sections of the NT transfected at E2 with the indicated constructs, harvested at E4 and immunostained for N-Cadherin (red); and for Sox2 (red) and HuCD (blue) to label progenitors and neurons, respectively. Transfection is reported by GFP expression. Summary: Schematic of the effects observed on tissue integrity. Gray cells correspond to electroporated cells. Scale bar represents 50 μm. See also S4 Fig. ΔMaml1, dominant-negative Mastermind-like 1; E, embryonic day; EP, electroporation; GFP, green fluorescent protein; HuCD, neuron-specific RNA-binding proteins HuC and HuD; N, neuron; Neurog2, Neurogenin 2; NT, neural tube; P, progenitor; RII-C1, Shroom3 binding site on ROCK2; Shroom3, Shroom family member 3; Sox2, SRY (sex determining region Y) box 2.
Fig 4.
Mib1 blocks the ability of Dll1 to Cis-inhibit Notch signaling.
(A) Top: Transverse sections of the NT transfected at E2, with the indicated constructs and harvested at E4. Immunostaining for Sox2 (blue) and HuCD (green) labels progenitors and neurons, respectively. Transfection is reported by H2B-Cherry expression. Arrowheads indicate ectopic Sox2+ progenitors adjacent to HuCD+ transfected neurons. Bottom: Summaries of the effects of Dll1 and Mib1 on neurogenesis. Red and gray cells correspond to electroporated (ep) and non-electroporated cells, respectively. Round and star-shaped cells correspond to progenitors and neurons, respectively. Blue outlines indicate cells changing fate, autonomously or non-autonomously, in each condition. (B, D) Quantification of the Hes5-VNP signal intensity in HuCD− cells either (B) non-transfected (surrounded by at least four transfected cells) or (D) transfected 24 hae with the indicated constructs. Data represent fold change compared to control. (B) n = 54, 35, 35, 54 cells were analyzed for control, Dll1, Dll1+Mib1, and Dll1+mbMib1, respectively. (D) n = 58, 59, 59, 66 cells were analyzed for control, Dll1, Dll1+Mib1, and Dll1+mbMib1, respectively. Data were collected from four to six embryos for each experimental group. ns, p > 0.05; **p < 0.01; ***p < 0.001 (Kruskal-Wallis test). (C, E) Quantification of the differentiation rate in (C) non-transfected neighbors (number of non-transfected HuCD+ cells adjacent to a HuCD+ transfected cell on the total number of adjacent cells) or (E) transfected cells (number of HuCD+ cells on total transfected cells) 48 hae with the indicated constructs. Data represent mean + SEM. n = 14 (6 embryos), 10 (8 embryos), 14 (6 embryos), 18 (6 embryos) sections were analyzed for control, Dll1, Dll1+Mib1, and Dll1+mbMib1, respectively. ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA). Analyses were performed on the same sections for (B) and (D), and for (C) and (E). Underlying data are provided in S1 Data. Scale bar represents 50 μm. See also S5 Fig. Ct, control; Dll1, Delta-like 1; E, embryonic day; ep, electroporated; hae, hour after electroporation; Hes5, Hairy and Enhancer of Split 5; HuCD, neuron-specific RNA-binding proteins HuC and HuD; H2B-Cherry, Histone 2B fused to Cherry; mbMib1, Mib1 constitutively tethered to the plasma membrane; Mib1, Mindbomb1; ns, nonsignificant; NT, neural tube; Sox2, SRY (sex determining region Y) box 2; VNP, Venus-NLS-PEST.
Fig 5.
Mib1 blocks Notch Cis-inhibition to defer differentiation and preserve neuroepithelial integrity.
(A) Quantification of Hes5-VNP intensity in HuCD− cells transfected with the indicated constructs at E2 and harvested 24 hae. Data represent fold change compared to control. A minimum of 108 cells were analyzed for each group. ns, p > 0.05; ***p < 0.001 (Kruskal-Wallis test). (B) Quantification of the differentiation rate (number of HuCD+ cells on total transfected cells). Data represent mean + SEM. n = 12 (3 embryos), 13 (6 embryos), 15 (6 embryos), 12 (4 embryos) sections were analyzed for control, Dll1, Dll1+ΔMib1, and ΔMib1, respectively. ns, p > 0.05; ***p < 0.001 (one-way ANOVA). (C) Quantification of the Hes5-VNP signal intensity in HuCD− and isolated cells transfected at E3 with the indicated constructs at low voltage (15 V) and harvested 8 h later. Data represent fold change compared to control. n = 37 (3 embryos), 31 (7 embryos), 15 (3 embryos), and 25 (4 embryos) cells were analyzed for control, ΔMib1, Neurog2, and Neurog2+ΔMib1, respectively. **p < 0.01 (Mann-Whitney U test). (D) Left: Transverse sections of the NT transfected at E2 with the indicated constructs, harvested at E3 and immunostained for HuCD (green) to label neurons. Transfection is reported by H2B-Cherry expression. Right: Quantification of the differentiation rate (number of HuCD+ cells on total transfected cells). Data represent mean + SEM. For 24 hae, n = 14, 12, 19, 7 sections collected from six embryos for each experimental group were analyzed for control, Neurog2, Neurog2+ΔMib1, and ΔMib1, respectively. ***p < 0.001 (one-way ANOVA). Underlying data are provided in S1 Data. (E, F) Transverse sections of the NT transfected at E2 with the indicated constructs and immunostained for N-Cadherin (red); and for Sox2 (red) and HuCD (blue) to label progenitors and neurons, respectively. Transfection is reported by H2B-Cherry expression (green). Scale bar represents 50 μm. ΔMib1, dominant-negative Mib1; Ct, control; Dll1, Delta-like 1; E, embryonic day; EP, electroporation; hae, hour after electroporation; Hes5, Hairy and Enchancer of Split 5; HuCD, neuron-specific RNA-binding proteins HuC and HuD; H2B-Cherry, Histone 2B fused to Cherry; Mib1, Mindbomb1; Neurog2, Neurogenin 2; ns, nonsignificant; NT, neural tube; Shroom3, shroom family member 3; Sox2, Sox2, SRY (sex determining region Y) box 2; VNP, Venus-NLS-PEST.
Fig 6.
Model for the role of Mib1-dependent Notch activity in the regulation of neuronal delamination.
Top: Prospective neurons maintain a high level of Notch activity until they fully differentiate. Mib1 is required during that transition phase to keep Dll1 from Cis-inhibiting the Notch receptor. This allows Notch to be Trans-activated by Dll1 present on neighboring cells (not represented here), resulting in the release of the NICD. When the Dll1/Mib1 ratio is sufficiently high, Cis-inhibition takes place and Notch activity is rapidly turned off. Bottom: Sustained Notch activity allows prospective neurons to shrink their apical area and keeps them from differentiating. As Notch activity is decreased, N-Cadherin levels are down-regulated and neuronal differentiation markers start being expressed. Dll1, Delta-like 1; Mib1, Mindbomb1; NICD, Notch intracellular domain; T, time.