Role of YAP in early ectodermal specification and a Huntington's Disease model of human neurulation
- Laboratory of of Stem Cell Biology and Molecular Embryology, The Rockefeller University, United States
- Howard Hughes Medical Institute, The Rockefeller University, United States
- Laboratory of Sensory Neuroscience, The Rockefeller University, United States
- ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal
- INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal
- Bioinformatics Resource Center, The Rockefeller University, United States
Figures
Figure 1 with 11 supplements
Dynamics of YAP expression and localization in early neuruloids.
(A) During the first 4 days of the differentiation protocol, pluripotent hESCs seeded on a micropattern undergo 3 days of neural induction (SB +LDN) followed by BMP4 induction of neurulation (SB …
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Figure 1—source data 1
Raw images for immunoblot shown in Figure 1D.
- https://cdn.elifesciences.org/articles/73075/elife-73075-fig1-data1-v1.zip
Figure 1—figure supplement 1
Screening of WT and HD YAP-GFP reporter lines.
(A) Genotypic screening of hESC clones from WT and HD backgrounds with PCR oligonucleotides (half-arrows) shows that multiple colonies underwent biallelic insertion of the GFP sequence. (B) …
Figure 1—figure supplement 2
Validation of WT and HD YAP-GFP reporter lines.
(Right) Fluorescent images of living WT and HD hESC clones expressing endogenous YAP-GFP and H2b-Cherry show the effect on YAP nuclear accumulation of cellular density and of treatment with 10 μM …
Figure 1—figure supplement 3
BMP4-dependent upregulation of YAP in D4 neuruloids.
(A) Fluorescent images of living D4 WT neuruloids demonstrate that the upregulation of YAP at the edge of the colony depends on BMP4 stimulation. (Left) Representative images show D4 neuruloids …
Figure 1—figure supplement 4
Quantification of immunoblots of total YAP.
(A) Quantification of protein expression quantification of two splicing alternatives of YAP (from D0 to D4, relative to Figure 1D) shows that both YAP proteins are downregulated upon neural …
Figure 1—figure supplement 5
Magnified images of D4 neuruloids labeled for YAP and DAPI.
Confocal microscopy of D4 neuruloids illustrates that YAP occurs at the edge of the colony. YAP can be found sporadically in the nuclei of WT cells and more frequently in WT cells treated with TRULI …
Figure 1—video 1
Live YAP dynamics in D3 WT neuruloid upon BMP4 stimulation.
Live fluorescent images of a quadrant of a D3 WT neuruloid, acquired in a time course during the first 24 hr following BMP4 administration (D4), show that YAP (YAP-GFP, green) is upregulated at the …
Figure 1—video 2
Live YAP dynamics in D3 WT neuruloid upon BMP4 stimulation (detail, YAP-GFP and H2b-mCherry).
Live fluorescent images of a portion of a D3 WT neuruloid, acquired in a time course between 18 hr and 24 hr following BMP4 administration (D4), show in more details that YAP (YAP-GFP, green) is …
Figure 1—video 3
Live YAP dynamics in D3 WT neuruloid upon BMP4 stimulation (detail, YAP-GFP).
Live fluorescent images of a portion of a D3 WT neuruloid, acquired in a time course between 18 hr and 24 hr following BMP4 administration (D4), show in more details that YAP (YAP-GFP, green) is …
Figure 1—video 4
Live YAP dynamics in D3 WT neuruloid upon BMP4 stimulation and activation by TRULI.
Live fluorescent images of a quadrant of a D3 WT neuruloid, treated with TRULI, acquired in a time course during the first 24 hr following BMP4 administration (D4) show that YAP (YAP-GFP, green) is …
Figure 1—video 5
Live YAP dynamics in D3 WT neuruloid upon BMP4 stimulation and activation by TRULI (Detail, YAP-GFP and H2b-mCherry).
Live fluorescent images of a portion of a D3 WT neuruloid, treated with TRULI, acquired in a time course between 18 hr and 24 hr following BMP4 administration (D4) show in more details that YAP …
Figure 1—video 6
Live YAP dynamics in D3 WT Neuruloid upon BMP4 stimulation and activation by TRULI (Detail, YAP-GFP).
Live fluorescent images of a portion of a D3 WT neuruloid, treated with TRULI, acquired in a time course between 18 hr and 24 hr following BMP4 administration (D4) show in more details that YAP …
Figure 2 with 1 supplement
Dynamics of YAP expression and localization in late neuruloids.
(A) In a schematic side view of the final phase of neuruloid formation, neural ectoderm (NE, dark blue) at the center is surrounded by nonneural ectoderm (NNE, cyan), which subsequently …
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Figure 2—source data 1
Raw images for immunoblot shown in Figure 2B.
- https://cdn.elifesciences.org/articles/73075/elife-73075-fig2-data1-v1.zip
Figure 2—figure supplement 1
Dynamics of YAP expression and localization in late neuruloids (magnified).
Representative magnified images of the late phase of neuruloid formation, D4–D6, show that YAP is expressed by SOX10+ (red) and PAX6+ (blue) at the different time points, but in these two lineages, …
Figure 3 with 1 supplement
Effect of sustained YAP activation on neural crest and neural epithelium.
Immunofluorescence images of D7 WT neuruloids demonstrate that treatment with 10 µM TRULI enhances the expression of neural ectoderm marked by PAX6 and of epidermis marked by KRT18 at the expense of …
Figure 3—figure supplement 1
Effect of sustained YAP activation on differentiation of neural crest (NC) and epidermis.
(A) Immunofluorescence images of hESCs after 10 days of a differentiation protocol for NC without (control) or with 10 μM TRULI show that treatment precludes the formation of NC (SOX10, red) and …
Figure 4 with 6 supplements
Dysregulation of YAP localization in HD neuruloids.
(A) Fluorescent images of the same portions of living neuruloids before (D3) and 24 hr after BMP4 administration (D4) show increased nuclear YAP in HD than in WT neuruloids. (B) Top, YAP …
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Figure 4—source data 1
Raw images for immunoblot shown in Figure 4C.
- https://cdn.elifesciences.org/articles/73075/elife-73075-fig4-data1-v1.zip
Figure 4—figure supplement 1
Dynamics of YAP localization in early WT and HD neuruloids.
(A) During the first 4 days of the differentiation protocol, pluripotent hESCs seeded on a micropattern undergo 3 days of neural induction (SB +LDN) followed by BMP4 induction of neurulation. (B) …
Figure 4—figure supplement 2
Induction of NNE in WT and HD D4 neuruloids.
Immunofluorescence images of D4 neuruloids demonstrate enhanced expression of TFAP2A at the perimeters of HD colonies in three independent experiments. At the bottom, the radial distribution of the …
Figure 4—figure supplement 3
Effect of YAP activation and HD mutation of rate of cell division in neuruloids.
Confocal images of D4–D7 neuruloids, labeled for phospho-histone H3 (pH3) to identify mitotic nuclei, show that YAP activation by TRULI or HD mutation induces an increase in the rate of cell …
Figure 4—video 1
Live YAP dynamics in D3 HD neuruloid upon BMP4 stimulation.
Live fluorescent images of a quadrant of a D3 HD neuruloid, acquired in a time course during the first 24 hr following BMP4 administration (D4), show that YAP (YAP-GFP, green) is upregulated at the …
Figure 4—video 2
Live YAP dynamics in D3 HD neuruloid upon BMP4 stimulation (Detail, YAP-GFP and H2b-mCherry).
Live fluorescent images of a portion of a D3 HD neuruloid, acquired in a time course between 18 hr and 24 hr following BMP4 administration (D4), show in more details that YAP (YAP-GFP, green) is …
Figure 4—video 3
Live YAP dynamics in D3 HD neuruloid upon BMP4 stimulation (Detail, YAP-GFP).
Live fluorescent images of a portion of a D3 HD neuruloid, acquired in a time course between 18 hr and 24 hr following BMP4 administration (D4), show in more details that YAP (YAP-GFP, green) is …
Figure 5 with 7 supplements
Expression of YAP target genes in WT and HD neuruloids.
(A) Violin plots show the expression of YAP and three representative target genes in the three clusters representing the main ectodermal lineages from scRNA-seq analysis of D4 neuruloids. Expression …
Figure 5—figure supplement 1
Ectodermal lineage annotation in scRNA-seq from D4 neuruloids.
(A) Uniform manifold approximation and projection (UMAP) shows the expression of key ectodermal lineage makers: PAX6 identifies neural ectoderm (NE) and TFAP2A delineates nonneural ectoderm (NNE). …
Figure 5—figure supplement 2
Marker genes for the three main human ectodermal lineages.
(Top) A heatmap displays the 100 genes that are most significantly expressed differentially between the three lineages. (Bottom) An enlargement displays the 20 genes that are most significantly …
Figure 5—figure supplement 3
Expression of key components of the Hippo pathway in D4 neuruloids.
(A) Violin plots document the expression of components of the Hippo pathway in the three ectodermal lineages. (B) A table summarizes the statistics for the pairwise differential-expression analysis …
Figure 5—figure supplement 4
Expression of YAP targets in the three human ectodermal lineages at D4.
(Left) A heatmap displays the expression of a large set of genes that are direct targets of YAP (Zanconato et al., 2015). (Right) The 100 genes most differentially expressed in epidermis show that …
Figure 5—figure supplement 5
Statistical analysis of D4 neuruloids.
Figure 5—figure supplement 6
Expression of YAP target genes in D7 WT and HD neuruloids.
(A) Uniform manifold approximation and projection (UMAP) shows the distribution of the three major ectodermal lineages in D7 neuruloid: neural ectoderm (NE, blue), neural crest (NC, red), and …
Figure 5—figure supplement 7
Statistical analysis of D7 neuruloids.
The statistics refer to the violin plots of Figure 5—figure supplement 6.
Figure 6 with 1 supplement
Perturbation of YAP activity in WT and HD neuruloids.
(A) Immunofluorescence images of D7 neuruloids show that the expansion of NE (PAX6, blue) at the expense of NC (SOX10, red) after exposure to 10 µM TRULI or in HD cells. Treatment with 0.3 µM …
Figure 6—figure supplement 1
Perturbation of YAP activity in WT and HD neuruloids.
(A) Immunofluorescence images of D7 neuruloids show that treatment with 0.3 µM verteporfin has no effect on WT colonies, whereas treatment with 10 µM TRULI results in the expansion of the central …
