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Boster Bio primary rabbit antibody to hoxc6
Primary Rabbit Antibody To Hoxc6, supplied by Boster Bio, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+hoxc6+antibody/Anti-HOXC6+Antibody/pm31629025-71-44-53
Average 90 stars, based on 2 article reviews

primary rabbit antibody to hoxc6 - by Bioz Stars, 2026-09

90/100 stars

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Incubation:

Article Title: Retraction
Article Snippet: .. The slices were incubated with 100 μL 5% bovine 145 serum albumin (BSA) blocking solution at 37°C for 30 min, followed by an incubation with 100 μL 146 primary rabbit-HOXC6 antibody (1 : 50, PB0948, Wuhan Boster Biological Technology, Ltd., 147 Wuhan, Hubei, China) overnight at 4°C. .. Following three PBS washes (3 min each time), the slices 148 were then incubated with biotinylated goat anti-rabbit antibody (1 : 100, HY90046, Shanghai 149 HengYuan Biological Technology Co., Ltd., Shanghai, China) at 37°C for 30 min. After PBS 150 washing, the slices were incubated with streptavidin-peroxidase solution (Beijing Zhongshan 151 Biological Technology Co., Ltd., Beijing, China) at 37°C for 30 min and developed by 152 diaminobenzidine staining solution (Beijing Bioss Biological Technology Co., Ltd., Beijing, China) 153 at room temperature.

Blocking Assay:

Image Search Results

Journal: Translational Oncology

Article Title: Comprehensive analysis and experimental verification of the mechanism of action of T cell-mediated tumor-killing related genes in Colon adenocarcinoma

doi: 10.1016/j.tranon.2024.101918

Figure Lengend Snippet: The target sequences for NPM1 siRNA.

Article Snippet: The tissue sections through deparaffinization and dehydration were incubated with polyclonal rabbit anti-human HOXC6 antibodies (1:500, Abcam, ab151575) overnight at 4 °C after epitope retrieval, H2O2 treatment, and non-specific antigens blocking.

Techniques: Sequencing

A list of the sequences of primer pairs for target genes.

Journal: Translational Oncology

Article Title: Comprehensive analysis and experimental verification of the mechanism of action of T cell-mediated tumor-killing related genes in Colon adenocarcinoma

doi: 10.1016/j.tranon.2024.101918

Figure Lengend Snippet: A list of the sequences of primer pairs for target genes.

Techniques: Sequencing

HOXC6 is elevated in colon cancer tissues. (A-E) The expression of C11orf96, CXCL9, HOXC6, VISG4 and CXCL13 in NCM460, SW480 and SW620 was detected by qRT-PCR and analyzed for relative quantification. (F-G) The expression of HOXC6 in cancer and paracancer tissues was detected and quantified using IHC. (H) The inhibition efficiency of small interfering RNAs was detected and analyzed for relative quantification in SW480 cell line. (I) Cell viability of SW480 was assayed after inhibition of HOXC6 expression in SW480 cell line. (J) The inhibition efficiency of small interfering RNAs was detected and analyzed for relative quantification in SW620 cell line. (K) Cell viability of SW480 was assayed after inhibition of HOXC6 expression in SW620 cell line. N = 3. *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001. N = 6/3, The results are presented as mean ± SD.

Journal: Translational Oncology

Article Title: Comprehensive analysis and experimental verification of the mechanism of action of T cell-mediated tumor-killing related genes in Colon adenocarcinoma

doi: 10.1016/j.tranon.2024.101918

Figure Lengend Snippet: HOXC6 is elevated in colon cancer tissues. (A-E) The expression of C11orf96, CXCL9, HOXC6, VISG4 and CXCL13 in NCM460, SW480 and SW620 was detected by qRT-PCR and analyzed for relative quantification. (F-G) The expression of HOXC6 in cancer and paracancer tissues was detected and quantified using IHC. (H) The inhibition efficiency of small interfering RNAs was detected and analyzed for relative quantification in SW480 cell line. (I) Cell viability of SW480 was assayed after inhibition of HOXC6 expression in SW480 cell line. (J) The inhibition efficiency of small interfering RNAs was detected and analyzed for relative quantification in SW620 cell line. (K) Cell viability of SW480 was assayed after inhibition of HOXC6 expression in SW620 cell line. N = 3. *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001. N = 6/3, The results are presented as mean ± SD.

Techniques: Expressing, Quantitative RT-PCR, Inhibition

HOXC6 promotes invasion and immunosuppression of colon cancer cell lines. (A-B) Alterations in cell migration as well as invasive capacity after inhibition of HOXC6 expression in SW480 and SW620 cell lines. (C-G) The expression levels of CXCL1, CXCL8, IL1β, IL4 and IL10 in SW480 cell line were detected by qRT-PCR before and after HOXC6 inhibition. (H-L) The expression levels of CXCL1, CXCL8, IL1β, IL4 and IL10 in SW620 cell line were detected by qRT-PCR before and after HOXC6 inhibition. N = 3. *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001. N = 6/3, The results are presented as mean ± SD.

Journal: Translational Oncology

Article Title: Comprehensive analysis and experimental verification of the mechanism of action of T cell-mediated tumor-killing related genes in Colon adenocarcinoma

doi: 10.1016/j.tranon.2024.101918

Figure Lengend Snippet: HOXC6 promotes invasion and immunosuppression of colon cancer cell lines. (A-B) Alterations in cell migration as well as invasive capacity after inhibition of HOXC6 expression in SW480 and SW620 cell lines. (C-G) The expression levels of CXCL1, CXCL8, IL1β, IL4 and IL10 in SW480 cell line were detected by qRT-PCR before and after HOXC6 inhibition. (H-L) The expression levels of CXCL1, CXCL8, IL1β, IL4 and IL10 in SW620 cell line were detected by qRT-PCR before and after HOXC6 inhibition. N = 3. *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001. N = 6/3, The results are presented as mean ± SD.

Techniques: Migration, Inhibition, Expressing, Quantitative RT-PCR

The Significant Changes in HOXC6 Expression at the Transcription Level between Different Types of Brain and CNS Cancers vs. Normal Brain Tissues (Oncomine Database)

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: The Significant Changes in HOXC6 Expression at the Transcription Level between Different Types of Brain and CNS Cancers vs. Normal Brain Tissues (Oncomine Database)

Article Snippet: Tissue slides were incubated with rabbit anti-HOXC6 antibody (1:500, Santa Cruz, sc-376330, America), anti-rabbit secondary antibody from Zymed Systems (InvitrogenCA) and 3,3’-diaminobenzidine to visualize IHC labelling.

Techniques: Expressing

HOXC6 was overexpressed in gliomas and correlated with clinical features. A Representative IHC staining of HOXC6 in WHO grade I-IV gliomas. B Mean density of HOXC6 in gliomas with different WHO grades. C - E High HOXC6 expression was positively correlated with high-grade gliomas in the TCGA, CGGA and Rembrandt datasets. F - G HOXC6 was significantly overexpressed in wild-type (WT) IDH gliomas based on the TCGA and CGGA datasets. H - I HOXC6 was upregulated in the 1p/19q non-codeletion group based on the TCGA and CGGA datasets. (* mean p < 0.05, ** mean p < 0.01, *** mean p < 0.001, **** mean p < 0.0001)

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: HOXC6 was overexpressed in gliomas and correlated with clinical features. A Representative IHC staining of HOXC6 in WHO grade I-IV gliomas. B Mean density of HOXC6 in gliomas with different WHO grades. C - E High HOXC6 expression was positively correlated with high-grade gliomas in the TCGA, CGGA and Rembrandt datasets. F - G HOXC6 was significantly overexpressed in wild-type (WT) IDH gliomas based on the TCGA and CGGA datasets. H - I HOXC6 was upregulated in the 1p/19q non-codeletion group based on the TCGA and CGGA datasets. (* mean p < 0.05, ** mean p < 0.01, *** mean p < 0.001, **** mean p < 0.0001)

Techniques: Immunohistochemistry, Expressing

HOXC6 predicts a poor prognosis in glioma patients. A – D Higher HOXC6 expression portended shorter OS in patients with gliomas based on clinical samples (n = 176) and the TCGA (n = 695), CGGA (n = 656) and Rembrandt datasets (n = 397). E – H ROC curves based on the above samples. I, Nomogram for predicting 2-, 3- and 5-year survival in glioma patients based on the TCGA dataset. J – K Calibration curves were used to predict the 2-, 3-, and 5-year survival in the TCGA and CGGA datasets

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: HOXC6 predicts a poor prognosis in glioma patients. A – D Higher HOXC6 expression portended shorter OS in patients with gliomas based on clinical samples (n = 176) and the TCGA (n = 695), CGGA (n = 656) and Rembrandt datasets (n = 397). E – H ROC curves based on the above samples. I, Nomogram for predicting 2-, 3- and 5-year survival in glioma patients based on the TCGA dataset. J – K Calibration curves were used to predict the 2-, 3-, and 5-year survival in the TCGA and CGGA datasets

Techniques: Expressing

Univariate and multivariate analyses of overall survival based on TCGA data

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: Univariate and multivariate analyses of overall survival based on TCGA data

Techniques:

HOXC6 promotes proliferation of glioma cells. A , B Western blot and RT q-PCR analysis of HOXC6 expression in various types of glioma cells as indicated. GAPDH was used as an internal control. C , D Verification of HOXC6 knockdown in U87 and U251 cells by Western blotting and q-PCR. E , F A CCK-8 assay was performed in U87 and U251 cells transfected with control shRNA and HOXC6 shRNA (n = 3, p < 0.05). G , H Colony-formation assays conducted in U87 and U251 cells transfected with control shRNA and HOXC6 shRNA (n = 3, p < 0.05). I , J EdU assays were performed in U87 and U251 cells transfected with control shRNA and HOXC6 shRNA (n = 3, p < 0.05). (* mean p < 0.05, ** mean p < 0.01, *** mean p < 0.001, **** mean p < 0.0001)

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: HOXC6 promotes proliferation of glioma cells. A , B Western blot and RT q-PCR analysis of HOXC6 expression in various types of glioma cells as indicated. GAPDH was used as an internal control. C , D Verification of HOXC6 knockdown in U87 and U251 cells by Western blotting and q-PCR. E , F A CCK-8 assay was performed in U87 and U251 cells transfected with control shRNA and HOXC6 shRNA (n = 3, p < 0.05). G , H Colony-formation assays conducted in U87 and U251 cells transfected with control shRNA and HOXC6 shRNA (n = 3, p < 0.05). I , J EdU assays were performed in U87 and U251 cells transfected with control shRNA and HOXC6 shRNA (n = 3, p < 0.05). (* mean p < 0.05, ** mean p < 0.01, *** mean p < 0.001, **** mean p < 0.0001)

Techniques: Western Blot, Expressing, Control, Knockdown, CCK-8 Assay, Transfection, shRNA

HOXC6-related biological processes in gliomas. A Conserved motifs identified by HOXC6 ChIP-seq. B Distribution of HOXC6 loci across the genome. C Representative GO analysis results. D Venn diagram for HOXC6 target genes from ChIP-seq and positive HOXC6-related genes in the TCGA and CGGA databases (Spearman’s R > 0.3, p < 0.05). E Network diagram of enrichment map results

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: HOXC6-related biological processes in gliomas. A Conserved motifs identified by HOXC6 ChIP-seq. B Distribution of HOXC6 loci across the genome. C Representative GO analysis results. D Venn diagram for HOXC6 target genes from ChIP-seq and positive HOXC6-related genes in the TCGA and CGGA databases (Spearman’s R > 0.3, p < 0.05). E Network diagram of enrichment map results

Techniques: ChIP-sequencing

HOXC6 promotes EMT processes. A Ridge plot to verify the gene signatures. B Venn diagram indicating the intersection of genes in RNA-seq and ChIP-seq. C Heatmap cluster based on the ChIPseq annotated targets differentially expressed in the sequencing results at the thresholds | fold change | > 2 and p value < 0.05. D Circular heatmap showing Spearman’s correlation between HOXC6 expression and the expression of EMT-related biomarkers based on the RNA-seq, TCGA, CGGA and Rembrandt databases

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: HOXC6 promotes EMT processes. A Ridge plot to verify the gene signatures. B Venn diagram indicating the intersection of genes in RNA-seq and ChIP-seq. C Heatmap cluster based on the ChIPseq annotated targets differentially expressed in the sequencing results at the thresholds | fold change | > 2 and p value < 0.05. D Circular heatmap showing Spearman’s correlation between HOXC6 expression and the expression of EMT-related biomarkers based on the RNA-seq, TCGA, CGGA and Rembrandt databases

Techniques: RNA Sequencing, ChIP-sequencing, Sequencing, Expressing

HOXC6 is involved in regulation of the tumour immune microenvironment. A HOXC6 expression is negatively related to tumour purity. B The expression of HOXC6 is positively related to the stromal score. C HOXC6 expression is positively related to the immune score. D - E The heatmap and the lollipop chart based on the TCGA and CGGA databases illustrate the relationship between HOXC6 and 28 infiltrating immune cell populations. F - G The heatmap and the radar chart based on the TCGA and CGGA databases illustrate the relationship between HOXC6 and immune checkpoint markers

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: HOXC6 is involved in regulation of the tumour immune microenvironment. A HOXC6 expression is negatively related to tumour purity. B The expression of HOXC6 is positively related to the stromal score. C HOXC6 expression is positively related to the immune score. D - E The heatmap and the lollipop chart based on the TCGA and CGGA databases illustrate the relationship between HOXC6 and 28 infiltrating immune cell populations. F - G The heatmap and the radar chart based on the TCGA and CGGA databases illustrate the relationship between HOXC6 and immune checkpoint markers

Techniques: Expressing

Verification of the positive correlation between HOXC6 expression and the mentioned genes. A The RT q-PCR analysis of HOXC6-correlated EMT-associated genes. B RT q-PCR analysis of HOXC6-correlated immune checkpoint proteins

Journal: Cancer Cell International

Article Title: HOXC6 impacts epithelial-mesenchymal transition and the immune microenvironment through gene transcription in gliomas

doi: 10.1186/s12935-022-02589-9

Figure Lengend Snippet: Verification of the positive correlation between HOXC6 expression and the mentioned genes. A The RT q-PCR analysis of HOXC6-correlated EMT-associated genes. B RT q-PCR analysis of HOXC6-correlated immune checkpoint proteins

Techniques: Expressing

(A) Identification of NP cell states using hierarchical clustering of gene expression profiles of the individual cells. (B) Cell state graph constructed from minimum spanning trees, color coded for the cell populations identified in Fig 2A. Stars indicate start and end cells for the reconstruction of transcriptional changes along pseudotime. Shading of edges between cells indicates how often the edge was used in the reconstruction of gene expression along pseudotime (see ). (C) Cell state graph color coded for expression levels of Irx3 , Olig2 , Ngn2 , Lhx3 , Isl1 , and Chat . (D) Inferred changes in gene expression over pseudotime from 9,000 shortest paths connecting start and end cells (stars in B). Each shortest path was resampled to a length of 41 pseudo–time points to enable statistical measurements of gene expression. Cell IDs are color coded according to cell states in (A). Quantification of the global rate of change in gene expression identifies three metastable states (light gray) separated by transition states, during which the rate of change in gene expression is increased (dark gray). Transition phases are defined as intervals along the pseudo-temporal timeline at which the second derivative of the global gene variation is negative, while metastable states are characterized by a positive second derivative. (E) Gene expression profiles along pseudo-time for NP TFs ( Irx3 , Pax6 , Nkx6 . 1 , and Olig2 ), genes associated with the transition to MNs ( Ngn2 , Lhx3 , and Neurod4 ) and MN markers ( Isl1/2 , Tubb3 , and Chat ). (F) Levels of gene expression for Hes1/5 , Olig2 , and Ngn2 over pseudotime. Note that Olig2 expression appears biphasic, with up-regulation of Olig2 concommitant to Ngn2 induction and repression of Hes1/5 in the transition phase from NP to MN. Chat , choline acetyltransferase ; KSP, K shortest paths; MN, motor neuron; NP, neural progenitor; pMN, MN progenitor; p3, V3 interneuron progenitor; Tubb3 , neuronal class III beta-tubulin.

Journal: PLoS Biology

Article Title: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

doi: 10.1371/journal.pbio.2003127

Figure Lengend Snippet: (A) Identification of NP cell states using hierarchical clustering of gene expression profiles of the individual cells. (B) Cell state graph constructed from minimum spanning trees, color coded for the cell populations identified in Fig 2A. Stars indicate start and end cells for the reconstruction of transcriptional changes along pseudotime. Shading of edges between cells indicates how often the edge was used in the reconstruction of gene expression along pseudotime (see ). (C) Cell state graph color coded for expression levels of Irx3 , Olig2 , Ngn2 , Lhx3 , Isl1 , and Chat . (D) Inferred changes in gene expression over pseudotime from 9,000 shortest paths connecting start and end cells (stars in B). Each shortest path was resampled to a length of 41 pseudo–time points to enable statistical measurements of gene expression. Cell IDs are color coded according to cell states in (A). Quantification of the global rate of change in gene expression identifies three metastable states (light gray) separated by transition states, during which the rate of change in gene expression is increased (dark gray). Transition phases are defined as intervals along the pseudo-temporal timeline at which the second derivative of the global gene variation is negative, while metastable states are characterized by a positive second derivative. (E) Gene expression profiles along pseudo-time for NP TFs ( Irx3 , Pax6 , Nkx6 . 1 , and Olig2 ), genes associated with the transition to MNs ( Ngn2 , Lhx3 , and Neurod4 ) and MN markers ( Isl1/2 , Tubb3 , and Chat ). (F) Levels of gene expression for Hes1/5 , Olig2 , and Ngn2 over pseudotime. Note that Olig2 expression appears biphasic, with up-regulation of Olig2 concommitant to Ngn2 induction and repression of Hes1/5 in the transition phase from NP to MN. Chat , choline acetyltransferase ; KSP, K shortest paths; MN, motor neuron; NP, neural progenitor; pMN, MN progenitor; p3, V3 interneuron progenitor; Tubb3 , neuronal class III beta-tubulin.

Article Snippet: Additional primary antibodies were used as follows: goat anti-β-galactosidase (Biogenesis 4600–1409 1:2,000), mouse anti-Cre (Covance MMS-106P, 1:2,000), rabbit anti-Dbx1 (kind gift of Susan Morten and Thomas Jessell, 1:8,000), rabbit anti-Fabp7 (Abcam ab32423, 1:2,000 or Chemical AB9558, 1:2,000), rat anti-FLAG (Stratagene 200474, 1:1,500), chicken anti-GFP (Abcam ab13970, 1:20,000), sheep anti-GFP (AbD Serotec 4745–1051, 1:800), rabbit anti-Hes1 ([ ], 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:250), mouse anti-Hb9/Mnx1 (DSHB, 1:40), mouse anti-Isl1/2 (DSHB, 1:100), goat anti-Isl1 (R&D AF1837, 1:1,000), rabbit anti-Lhx3 (Abcam ab14555, 1:500), mouse anti-NeuN (Rbfox3, Chemicon/Millipore MAB377, 1:1,000), rat anti-chick Neurod4 (NeuroM [ ]), goat anti-Ngn2 (Santa Cruz Biotechnology sc-19233, 1:500), mouse anti-Ngn2 (5C6, [ ], 1:50), guinea pig anti-chick Ngn2 ([ ], 1:32,000), mouse anti-Nkx2.2 (DSHB, 1:25), mouse anti-Nkx6.1 (DSHB, 1:100), rabbit anti-Olig2 (Millipore AB9610, 1:1,000), guinea pig anti-mouse Olig2 ([ ] 1:20,000), guinea pig anti-chick Olig2 ([ ], 1:8,000), rabbit anti-Pax6 (Millipore AB2237, 1:1,000), mouse anti-Pax6 (DSHB, 1:25), goat anti-Sox1 (R&D AF3369, 1:500), goat anti-Sox2 (Santa Cruz Biotechnology sc-17320, 1:2,000), rabbit anti-Sox2 ([ ], 1:2500), rabbit anti-TagRFP (Evrogen AB233, 1:1,000), rabbit anti-Tubb3 (Covance PRB-435P, 1:2,000), mouse anti-Tubb3 (Covance MMS-435P, 1:1,000), rabbit anti-Zbtb18 (Proteintech 12714-1-AP, 1:1,000).

Techniques: Gene Expression, Construct, Expressing

(A–D) Expression patterns of Ngn2 (green in A), Olig2 (red in A, C, D), Hes1 (red in B, green in C), and Hes5 (green in B, D) in the neural tube at e10.5. Note the low expression levels of Hes1/5 and high expression levels of Ngn2 in the pMN domain (compare A, B). (E) Hes5 (green) expression coincides with the expression of high levels of Pax6 (red) in the intermediate neural tube. (F, G) Hes1 expression (green) is readily detected in both Nkx2.2 + p3 progenitors (red in F) and floor plate cells labelled by Foxa2 expression (red in G). (H–Q′) Time course of Olig2 (blue), Hes1 (red), Hes5 (red), and Ngn2 (green) expression in neural tubes between e8.5 and e10.5. Multiple panels shown for e9.5 reflect developmental progression from caudal to rostral positions along the neuraxis. Hes1 expression appears to recede from the ventral neural tube upon the onset of Olig2 expression at e8.5 (H) and is thereafter absent from most Olig2+ cells (I–L). Olig2 and Hes5 are initially coexpressed (M, N). Over time, Hes5 expression progressively disappears from the pMN domain (N–Q), and Ngn2 concomitantly increases (N′–Q′). Insets show single channel images of the outlined area for the respective markers. Scale bars = 50 μm. e, embryonic day; pMN, MN progenitor; p3, V3 interneuron progenitor.

Journal: PLoS Biology

Article Title: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

doi: 10.1371/journal.pbio.2003127

Figure Lengend Snippet: (A–D) Expression patterns of Ngn2 (green in A), Olig2 (red in A, C, D), Hes1 (red in B, green in C), and Hes5 (green in B, D) in the neural tube at e10.5. Note the low expression levels of Hes1/5 and high expression levels of Ngn2 in the pMN domain (compare A, B). (E) Hes5 (green) expression coincides with the expression of high levels of Pax6 (red) in the intermediate neural tube. (F, G) Hes1 expression (green) is readily detected in both Nkx2.2 + p3 progenitors (red in F) and floor plate cells labelled by Foxa2 expression (red in G). (H–Q′) Time course of Olig2 (blue), Hes1 (red), Hes5 (red), and Ngn2 (green) expression in neural tubes between e8.5 and e10.5. Multiple panels shown for e9.5 reflect developmental progression from caudal to rostral positions along the neuraxis. Hes1 expression appears to recede from the ventral neural tube upon the onset of Olig2 expression at e8.5 (H) and is thereafter absent from most Olig2+ cells (I–L). Olig2 and Hes5 are initially coexpressed (M, N). Over time, Hes5 expression progressively disappears from the pMN domain (N–Q), and Ngn2 concomitantly increases (N′–Q′). Insets show single channel images of the outlined area for the respective markers. Scale bars = 50 μm. e, embryonic day; pMN, MN progenitor; p3, V3 interneuron progenitor.

Techniques: Expressing

(A–D) Expression of Cre (green in A–D), Olig2 (red in A), Ngn2 (red in B), Hes1 (red in C, grey in C′), and Hes5 (red in D, grey in D′) in e10.5 Olig2 Cre heterozygous embryos. (E–H) In Olig2 Cre/Cre homozygous mutants, Hes1 expands dorsally (G, G′) and Hes5 ventrally (H, H′) into the pMN domain, marked by Cre expressed from the Olig2 locus. The expansion of Hes1/5 coincides with a loss of the high levels of Ngn2 normally seen in the pMN domain. (I) Quantification of Hes1, Hes5, and Ngn2 expression in Olig2 Cre heterozygous and homozygous embryos. The overlap between Cre and Hes1/5 significantly increases in Olig2 Cre homozygotes, while overlap between Ngn2 and Cre is strongly reduced. Plot shows the mean ±SEM from multiple sections collected from 3–5 embryos for each group. Each section is represented by a single dot, with n = 8–11 for each group. Underlying data are provided in . **** p < 0.0001, unpaired t test. (J–S) Electroporation of myc-tagged OLIG2 and an OLIG2-bHLH-Engrailed repressor domain fusion protein in chick neural tubes represses expression of the Hes5 homologues HES5-1 – HES5-3 (K–M; Q–R) and the Hes1 homologue HAIRY1 (N, S). “+” indicates transfected side of the spinal cords. Results are representative of >5 successfully transfected embryos collected from two or more experiments. Scale bars = 50 μm. bHLH, basic helix-loop-helix DNA binding domain; Cre, bacteriophage P1 Cre recombinase; e, embryonic day; EnR, Engrailed transcriptional repression domain; EP, electroporation; pMN, MN progenitor.

Journal: PLoS Biology

Article Title: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

doi: 10.1371/journal.pbio.2003127

Figure Lengend Snippet: (A–D) Expression of Cre (green in A–D), Olig2 (red in A), Ngn2 (red in B), Hes1 (red in C, grey in C′), and Hes5 (red in D, grey in D′) in e10.5 Olig2 Cre heterozygous embryos. (E–H) In Olig2 Cre/Cre homozygous mutants, Hes1 expands dorsally (G, G′) and Hes5 ventrally (H, H′) into the pMN domain, marked by Cre expressed from the Olig2 locus. The expansion of Hes1/5 coincides with a loss of the high levels of Ngn2 normally seen in the pMN domain. (I) Quantification of Hes1, Hes5, and Ngn2 expression in Olig2 Cre heterozygous and homozygous embryos. The overlap between Cre and Hes1/5 significantly increases in Olig2 Cre homozygotes, while overlap between Ngn2 and Cre is strongly reduced. Plot shows the mean ±SEM from multiple sections collected from 3–5 embryos for each group. Each section is represented by a single dot, with n = 8–11 for each group. Underlying data are provided in . **** p < 0.0001, unpaired t test. (J–S) Electroporation of myc-tagged OLIG2 and an OLIG2-bHLH-Engrailed repressor domain fusion protein in chick neural tubes represses expression of the Hes5 homologues HES5-1 – HES5-3 (K–M; Q–R) and the Hes1 homologue HAIRY1 (N, S). “+” indicates transfected side of the spinal cords. Results are representative of >5 successfully transfected embryos collected from two or more experiments. Scale bars = 50 μm. bHLH, basic helix-loop-helix DNA binding domain; Cre, bacteriophage P1 Cre recombinase; e, embryonic day; EnR, Engrailed transcriptional repression domain; EP, electroporation; pMN, MN progenitor.

Techniques: Expressing, Electroporation, Transfection, Binding Assay

(A) Identification of an evolutionarily conserved element containing an E-box in the vicinity of the Hes5 genomic locus in chick, mouse, and human (Hes5(e1)). (B) Analysis of Olig2 Chip-Seq data from reveals Olig2 binding sites in the vicinity of the Hes1 and Hes5 genes. The peak corresponding to the Hes5(e1) element is highlighted in red. (C) Electrophoretic mobility shift assays show that both Olig2 and E12 homodimers can individually bind to the Hes5(e1) E-box and do not form any heterodimeric complexes (lanes 1–4). Positions of the different protein complexes are indicated by colored arrows. Binding depends on the E-box, as both proteins fail to bind probes containing an E-box mutation (Hes5(e1ΔE)) (lanes 5–7). Olig2 binding to Hes5(e1) can be abolished by the addition of unlabelled Hes5(e1) probes, but not those containing the E-box mutation (lanes 8–14). (D) Id1 inhibits binding of E12, but not of Olig2 or Ngn2, to the Hes5(e1) element. Olig2, E12, and Ngn2 alone or Ngn2/E12 heterodimers can bind the Hes5(e1) element. Mixing Olig2 or Ngn2 with Id1 does not inhibit their homodimeric binding activities (lanes 2, 5, 8, and 10). In contrast, Id1 strongly inhibits binding of both E12/E12 and Ngn2/E12 complexes (lanes 6 and 10). The addition of E12 without and with Id1 does not affect Olig2 binding efficiency (lanes 2, 4, and 7). ATG, translational initation codon; Chip-Seq, chromatin immunoprecipitation-sequence; E-box, bHLH transcription factor binding site; N2, Ngn2 protein; O2, Olig2 protein.

Journal: PLoS Biology

Article Title: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

doi: 10.1371/journal.pbio.2003127

Figure Lengend Snippet: (A) Identification of an evolutionarily conserved element containing an E-box in the vicinity of the Hes5 genomic locus in chick, mouse, and human (Hes5(e1)). (B) Analysis of Olig2 Chip-Seq data from reveals Olig2 binding sites in the vicinity of the Hes1 and Hes5 genes. The peak corresponding to the Hes5(e1) element is highlighted in red. (C) Electrophoretic mobility shift assays show that both Olig2 and E12 homodimers can individually bind to the Hes5(e1) E-box and do not form any heterodimeric complexes (lanes 1–4). Positions of the different protein complexes are indicated by colored arrows. Binding depends on the E-box, as both proteins fail to bind probes containing an E-box mutation (Hes5(e1ΔE)) (lanes 5–7). Olig2 binding to Hes5(e1) can be abolished by the addition of unlabelled Hes5(e1) probes, but not those containing the E-box mutation (lanes 8–14). (D) Id1 inhibits binding of E12, but not of Olig2 or Ngn2, to the Hes5(e1) element. Olig2, E12, and Ngn2 alone or Ngn2/E12 heterodimers can bind the Hes5(e1) element. Mixing Olig2 or Ngn2 with Id1 does not inhibit their homodimeric binding activities (lanes 2, 5, 8, and 10). In contrast, Id1 strongly inhibits binding of both E12/E12 and Ngn2/E12 complexes (lanes 6 and 10). The addition of E12 without and with Id1 does not affect Olig2 binding efficiency (lanes 2, 4, and 7). ATG, translational initation codon; Chip-Seq, chromatin immunoprecipitation-sequence; E-box, bHLH transcription factor binding site; N2, Ngn2 protein; O2, Olig2 protein.

Techniques: ChIP-sequencing, Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, Chromatin Immunoprecipitation, Sequencing

(A, B) Co-electroporation of CMV/β-actin::nLacZ and Hes5(e1) reporter plasmids into chick spinal cord. Although electroporation (revealed by β-Gal antibody staining, magenta in [A]) is uniform along the dorsal-ventral axis, expression of the EGFP reporter is confined to intermediate parts of the neural tube (A, B), and little coexpression of Olig2 and EGFP was detected (B). (C) Design of Hes5(e1) and Hes5(e1ΔE) reporters. The Hes5(e1) element was cloned in front of β-globin minimal promoter to drive EGFP reporter gene expression. To test the importance of the E-box in the Hes5(e1) element, critical base pairs for Olig2 binding were mutated (red). (D, E) Co-electroporation of CMV/β-actin::-nLacZ and Hes5(e1ΔE) reporter plasmids into chick spinal cord. In contrast to the Hes5(e1) reporter plasmid, significant coexpression of Olig2 and GFP in the pMN domain is detected (E). Note that E-box mutation reduced the basal activity of the reporter such that longer exposure times were needed to achieve the signal levels seen in the intermediate spinal cord with the nonmutated Hes5(e1) reporter . (F) Scatter dot plots display the dorsal-ventral positions (distance from the roof plate) of individual cells expressing the Hes5(e1) and Hes5(e1ΔE) reporters, relative to CMV/β-actin::-nLacZ and Olig2. Results are aggregated from five representative sections taken from five well-electroporated and stage-matched spinal cords. The Hes5(e1ΔE) reporter exhibits a significant ventral shift in its activity and considerable overlap with Olig2 expression (blue dotted box). Box plots include the median and whiskers represent 5th and 95th percentiles. Data points that lay outside the DV scale used to assess these experiments were excluded from this analysis. ** p = 0.0005, Mann-Whitney test; p = 0.6649. Underlying data are provided in . (G) EGFP expression in Hes5(e1)-nEGFP whole mount embryos at e10.5. (H–H″) Cryosections of Hes5(e1)-nEGFP embryos at e10.5 assayed for GFP, Olig2, and Hes5. EGFP expression colocalizes with Hes5 expression (H″) but not with Olig2 (H). (I–N) Hes5(e1)-nEGFP expression in Olig2 heterozygous (I, K, L) and homozygous mutants (J, M, N). In Olig2 heterozygotes, little nEGFP expression can be detected in the Olig2 expression domain, resulting in a pronounced gap between the expression domains of EGFP, Nkx2.2, and Hes1 (K, L). By contrast, the EGFP, Nkx2.2, and Hes1 expression domains directly abut each other in Olig2 homozygous mutants (M, N). β-Gal, beta-galactosidase; βGlob, beta-globin; CMV/β-actin::nLacZ, cytomegalovirus/chick beta-actin promoter driving nuclear LacZ gene expression; ΔE, E-box deletion; E-box, bHLH protein binding site; EGFP, enhanced green fluorescent protein; FP, floor plate; GFP, green fluorescent protein; H5(e1), Hes5(e1) genomic element; ns, not significant; pMN, motor neuron progenitor; WT, wild-type.

Journal: PLoS Biology

Article Title: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

doi: 10.1371/journal.pbio.2003127

Figure Lengend Snippet: (A, B) Co-electroporation of CMV/β-actin::nLacZ and Hes5(e1) reporter plasmids into chick spinal cord. Although electroporation (revealed by β-Gal antibody staining, magenta in [A]) is uniform along the dorsal-ventral axis, expression of the EGFP reporter is confined to intermediate parts of the neural tube (A, B), and little coexpression of Olig2 and EGFP was detected (B). (C) Design of Hes5(e1) and Hes5(e1ΔE) reporters. The Hes5(e1) element was cloned in front of β-globin minimal promoter to drive EGFP reporter gene expression. To test the importance of the E-box in the Hes5(e1) element, critical base pairs for Olig2 binding were mutated (red). (D, E) Co-electroporation of CMV/β-actin::-nLacZ and Hes5(e1ΔE) reporter plasmids into chick spinal cord. In contrast to the Hes5(e1) reporter plasmid, significant coexpression of Olig2 and GFP in the pMN domain is detected (E). Note that E-box mutation reduced the basal activity of the reporter such that longer exposure times were needed to achieve the signal levels seen in the intermediate spinal cord with the nonmutated Hes5(e1) reporter . (F) Scatter dot plots display the dorsal-ventral positions (distance from the roof plate) of individual cells expressing the Hes5(e1) and Hes5(e1ΔE) reporters, relative to CMV/β-actin::-nLacZ and Olig2. Results are aggregated from five representative sections taken from five well-electroporated and stage-matched spinal cords. The Hes5(e1ΔE) reporter exhibits a significant ventral shift in its activity and considerable overlap with Olig2 expression (blue dotted box). Box plots include the median and whiskers represent 5th and 95th percentiles. Data points that lay outside the DV scale used to assess these experiments were excluded from this analysis. ** p = 0.0005, Mann-Whitney test; p = 0.6649. Underlying data are provided in . (G) EGFP expression in Hes5(e1)-nEGFP whole mount embryos at e10.5. (H–H″) Cryosections of Hes5(e1)-nEGFP embryos at e10.5 assayed for GFP, Olig2, and Hes5. EGFP expression colocalizes with Hes5 expression (H″) but not with Olig2 (H). (I–N) Hes5(e1)-nEGFP expression in Olig2 heterozygous (I, K, L) and homozygous mutants (J, M, N). In Olig2 heterozygotes, little nEGFP expression can be detected in the Olig2 expression domain, resulting in a pronounced gap between the expression domains of EGFP, Nkx2.2, and Hes1 (K, L). By contrast, the EGFP, Nkx2.2, and Hes1 expression domains directly abut each other in Olig2 homozygous mutants (M, N). β-Gal, beta-galactosidase; βGlob, beta-globin; CMV/β-actin::nLacZ, cytomegalovirus/chick beta-actin promoter driving nuclear LacZ gene expression; ΔE, E-box deletion; E-box, bHLH protein binding site; EGFP, enhanced green fluorescent protein; FP, floor plate; GFP, green fluorescent protein; H5(e1), Hes5(e1) genomic element; ns, not significant; pMN, motor neuron progenitor; WT, wild-type.

Techniques: Electroporation, Staining, Expressing, Clone Assay, Gene Expression, Binding Assay, Plasmid Preparation, Mutagenesis, Activity Assay, MANN-WHITNEY, Protein Binding

(A) Proposed model of the Olig2-controlled gene regulatory network. Olig2 not only acts as central organizer for dorsal-ventral patterning in the spinal cord but also controls the rate of MN differentiation through direct repression of Hes TFs. This leads to a higher levels of Ngn2 expression and, consequently, a higher rate of neuronal differentiation in the pMN domain, compared to adjacent progenitor domains. (B) Olig2 is a core component of the Shh-controlled gene regulatory network that patterns the ventral spinal cord [ , ]. (C) Olig2-mediated down-regulation of the Notch effectors Hes1/5 relieves repression of Ngn2 in the pMN domain. (D) Consolidated activities of Ngn2 and Olig2 cause differentiation of NPs to MNs. Olig2 promotes differentiation of MNs through repression of alternative IN cell fates. bHLH, basic helix-loop-helix; IN, interneuron; MN, motor neuron; NP, neural progenitor; pMN, MN progenitor; p2, V2 interneuron progenitor; p3, V3 interneuron progenitor; RA, retinoic acid; Shh, sonic hedgehog; TF, transcription factor; V2, V2 interneuron; V3, V3 interneuron.

Journal: PLoS Biology

Article Title: Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

doi: 10.1371/journal.pbio.2003127

Figure Lengend Snippet: (A) Proposed model of the Olig2-controlled gene regulatory network. Olig2 not only acts as central organizer for dorsal-ventral patterning in the spinal cord but also controls the rate of MN differentiation through direct repression of Hes TFs. This leads to a higher levels of Ngn2 expression and, consequently, a higher rate of neuronal differentiation in the pMN domain, compared to adjacent progenitor domains. (B) Olig2 is a core component of the Shh-controlled gene regulatory network that patterns the ventral spinal cord [ , ]. (C) Olig2-mediated down-regulation of the Notch effectors Hes1/5 relieves repression of Ngn2 in the pMN domain. (D) Consolidated activities of Ngn2 and Olig2 cause differentiation of NPs to MNs. Olig2 promotes differentiation of MNs through repression of alternative IN cell fates. bHLH, basic helix-loop-helix; IN, interneuron; MN, motor neuron; NP, neural progenitor; pMN, MN progenitor; p2, V2 interneuron progenitor; p3, V3 interneuron progenitor; RA, retinoic acid; Shh, sonic hedgehog; TF, transcription factor; V2, V2 interneuron; V3, V3 interneuron.

Techniques: Expressing

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