Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A) Expression of Hoxc6 and FoxP1 in LMC neurons between e10.5 and e13.5. Images show ventral quadrant of the spinal cord and LMC neurons are identified by FoxP1 expression. Hoxc6 protein is detected in postmitotic MNs but not in MN progenitors (pMNs). At e10.5 Hoxc6 is expressed by FoxP1+ LMC neurons but by e12.5 is attenuated in most LMC neurons. (B) Grayscale images showing progressive decline in Hoxc6 protein expression from LMC neurons. (C–J) Expression of Hox4–Hox8 paralogs at brachial levels of the spinal cord. Hoxb6 and Hoxb7 are expressed in pMNs. (K) Summary of Hox expression patterns. Hoxc4, Hoxa5, Hoxc6 expression domains extend further rostrally in the spinal cord and are not shown. Hoxa7 and Hoxc8 expression also extends into thoracic levels, indicated in light grey.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A–D) Loss of LMC neurons at brachial levels of the spinal cord in Hoxa6/Hoxc6 mutants at e12.5. LMC neurons are defined by FoxP1 and Raldh2 expression. The most pronounced losses are observed in rostral brachial regions (A, C), where the number of FoxP1 MNs is reduced and Raldh2 is only weakly detected in Hoxa6/Hoxc6 double mutants. (E–F) Increase in the number of Hb9+, Isl1+, Lhx3− HMC neurons in Hox6 mutants. Rostral brachial spinal cord also normally contains a small MN population that coexpresses Hb9 and Isl1, but for simplicity is not shown. The number of Lhx3+ MMC neurons is unchanged. (G–H) Summary of MN columnar defects in Hoxa6/c6 mutants. (I) Quantification of total number of LMC neurons in Hox6 mutants. Numbers are extrapolated from serial sections and are based on FoxP1 MN counts. Controls are averaged from Hoxa6 −/− Hoxc6+/+ , Hoxa6+/−Hoxc6+/− , and Hoxa6−/−, Hoxc6+/− embryos as the number of LMC neurons in these alleles was similar to wildtype embryos (see ). (J) Quantification of LMC loss in serial sections of Hoxa6/c6 mutants. Sections numbered 1–9 represent progression from rostral to caudal levels of the LMC.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A) Schematic of Hox patterns and Hox-dependent MN columnar subtypes at brachial and thoracic levels. Lateral motor column (LMC) neurons express high FoxP1 levels and Raldh2. Preganglionic column (PGC) neurons express low FoxP1 levels and pSmad. Model on right shows Hox interactions specifying LMC and PGC MN subtypes. (B–G) Effects of misexpression of Hox4–Hox8 genes at thoracic levels. Electroporated motor neurons are indicated by Hox protein+Isl1/2 costaining. (B) Hoxc4 fails to induce Foxp1 or Raldh2 expression and does not repress Hoxc9 . (C) Hoxa5 induces LMC identity, blocks pSmad, but fails to repress Hoxc9 . (D–E) Hoxa6 and Hoxc6 induce LMC MNs and effectively extinguish Hoxc9 and pSmad. (F–G) Hoxa7 and Hoxc8 induce LMC fate and display attenuated capacity to repress Hoxc9 . Hoxa7 and Hoxc8 electroporations are shown at caudal thoracic levels, where endogenous expression is minimal. Embryos were electroporated at HH st13–14 and analyzed two days later.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A) Serial sections from the caudal half of the LMC in Hoxc6 mutants showing loss of the Pea3+ pool at e12.5. Rostral to caudal sections are shown from left to right. Images are taken from a level similar to sections 1, 3, and 5 in panel D. Results are similar between Hoxc6 and Hoxa6/Hoxc6 double mutant sections. (B) Quantification of the loss of Pea3+ MNs in Hox6 mutants. Graph on left shows average Pea3+ MN number per section. Graph on right shows Pea3+ MN number as a percentage of the total LMC. (C) Expression of Scip+ LMC neurons in Hoxc6 mutants. Images are taken from a level similar to sections 3, 4, and 5 in panel D. (D) Quantification of Scip+ MNs in Hox6 mutants. Graph on left shows Scip+ MN number per section. Graph on right shows Scip MN number as a percentage of the total LMC.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Mutagenesis, Expressing

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A–D) Whole mount GFP staining in Hb9::GFP, Hoxc6 mutant and control mice. At both e12.5 and e13.5 there is a severe reduction in the innervation of the cutaneous maximus (cm) muscle (outlined with dashed green line). At e12.5 Hoxc6 mutants are characterized by an abnormal bifurcation at the distal end of the ulnar (uln) nerve, and at e13.5 there is an atypical misdirection of axons towards the medial portion of the paw (red arrowheads in A–D). Musculocutaneous nerve is indicated by white arrowheads in panels C and D. Similar results were obtained in n = 3 control and Hoxc6 mutants at both ages. (E) Retrograde labeling from the ulnar nerve in Hoxc6 mutants. In Hoxc6 mutants, many Scip− MNs are labeled with RhD. In addition there is a reduction in the number of Scip+ RhD+ labeled MNs in the Hoxc6 mutant. Section shown for the Hoxc6 mutant is rostral to the central portion of the Scip+ LMC pool, at a segmental level occupied predominantly by former Pea3+ MNs (See also ). Schematic summarizes the distribution of labeled MNs after tracer injection into the ulnar nerve in caudal (c) LMC neurons.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Staining, Mutagenesis, Control, Labeling, Injection

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A–B) Electroporation of Hoxc6 induces Pea3 expression at thoracic levels. (C–F) Expression of Hoxa7 and Hoxc8 at thoracic levels induces Foxp1 but not Pea3 expression. Similar results were obtained with Hoxa5 (data not shown). (G–H) A chimera of the Hoxc6 N-terminus (including the YPWM motif) and the Hoxc4 homeodomain (HD) induces high levels of Foxp1 expression. (I–J) The Hoxc6:c4 chimera fails to induce Pea3 expression at thoracic levels and shows an attenuated capacity to repress Hoxc9. (K–L) A chimera of the Hoxc6 N-terminus to the homeodomain of Hoxc8 induces LMC MNs, but fails to induce Pea3 expression. (M) Model for Hox interactions in MN subtype specification. During LMC specification multiple Hox paralogs converge on the regulation of pan-LMC genes such as FoxP1 and Raldh2. In motor neuron pools more specific Hox activities are employed.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Electroporation, Expressing

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A) Analysis of a Foxp1 enhancer transgene ( hs1149::lacZ ) expression in the spinal cord. This enhancer targets high levels of lacZ expression at limb-levels of the spinal cord, but low levels thoracically. Expression is upregulated in Hoxc9 mutants indicating it is under Hox control. Further analysis of this line however revealed that this enhancer targets a broad range of cell types (data not shown), suggesting that other elements are required to achieve MN expression and specificity. (B) Gel mobility shift assays with Hoxc6 and Pbx3 on a putative Hox site in hs1149 . (C) ChIP analysis of Hoxc6 binding to the hs1149 region in mouse spinal cord. (D) Binding of Hoxc6 mutant derivatives to hs1149 and an optimized Hox binding site ( Fkh250Con* ). Although Hoxc6NΔ91 shows reduced binding in this assay, it is as effective as wildtype Hoxc6 in inducing LMC fates at thoracic levels . Equivalent molar amounts of Hox protein (2 pmol) were used in each assay.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Expressing, Control, Mobility Shift, Binding Assay, Mutagenesis

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A) Model of the transcriptional network controlling MN subtype identities at brachial and thoracic levels of the spinal cord. Hoxc6 promotes LMC fate through induction of high levels of FoxP1 and Raldh2 expression and restricts Hoxc9 . Within the LMC a network of Hox4–Hox8 proteins controls pool fates. The network specifying the Pea3 pool is shown. Hoxc6 repression (RD) and activation domains (AD) are indicated. (B–F) Effects of expression of various Hoxc6 mutant derivatives on LMC specification at thoracic levels. Hoxc6 expression was monitored using either an HA antibody or an antibody against Hoxc6. Electroporated MNs are identified by co-staining with Isl1/2. (B) Hoxc6 activates Foxp1 , Raldh2 , Pea3 and represses Hoxc9 . (C) Deletion of the highly conserved “SYF” motif (Hoxc6NΔ13) does not affect Hoxc6 activity in MNs. (D–E) Hoxc6NΔ64 and Hoxc6NΔ104 induce LMC fate but fail to induce Pea3 or repress Hoxc9 . (F) Hoxc6NΔ111 fails to induce Foxp1 and Raldh2 or repress Hoxc9 .

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Expressing, Activation Assay, Mutagenesis, Staining, Activity Assay

Journal: PLoS Genetics

Article Title: Genetic and Functional Modularity of Hox Activities in the Specification of Limb-Innervating Motor Neurons

doi: 10.1371/journal.pgen.1003184

Figure Lengend Snippet: (A) Expression of Hoxc6NΔ91 at thoracic levels activates Foxp1 and Raldh2 , but fails to induce Pea3 or repress Hoxc9 . (B) Expression of Hoxc6IM (YPWM->AAAM mutation) at thoracic levels activates Foxp1 , Raldh2 , Pea3 and represses Hoxc9 , similar to wildtype Hoxc6. The ability of Hoxc6IM to activate Pea3 could reflect a Pbx-independent program that specifies the Pea3+ pool. (C) Expression of Hoxc6NΔ91+IM fails to repress Hoxc9 or induce LMC identity. (D) Interpretation of results. Hoxc6 normally induces LMC-specific genes in a Pbx-dependent manner, and contributes to the exclusion of Hoxc9 . When the Pbx interaction domain is mutated, Hoxc9 is still repressed. As Hoxc9 normally acts to dampen Foxp1 expression, the absence of Hoxc9 allows Hox proteins resident to the thoracic spinal cord to induce Foxp1 and activate the LMC program. When both the Pbx interaction motif and Hoxc9 repression domain are deleted, Hoxc9 is expressed, ensuring Foxp1 is not activated, and preventing LMC specification.

Article Snippet: Antibodies used were goat anti-mouse Hoxc6 (Santa Cruz) or rabbit ant-mouse Hoxc6 (Abcam).

Techniques: Expressing, Mutagenesis

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

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.

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

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.

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: Expressing, Quantitative RT-PCR, Inhibition

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.

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: Migration, Inhibition, Expressing, Quantitative RT-PCR

Journal: Neuron

Article Title: Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration

doi: 10.1016/j.neuron.2022.01.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used for motor neuron characterization include mouse anti-Islet-1/2 (DSHB, 1:500), mouse anti-HB9/Mnx1 (DSHB, 1:500), goat anti-SCIP (Santa Cruz, 1:5000), anti-HOXA5 ( Dasen et al., 2005 ), anti-FOXP1 ( Dasen et al., 2008 ), anti-HOXC6 ( Liu et al., 2001 ), anti-HOXC9 ( Jung et al., 2010 ), anti-LHX3 ( Tsuchida et al., 1994 ), rabbit anti-OLIG2 (Proteintech, 1:500), mouse anti-p-Histone H3 (Santa Cruz, 1:400), Alexa 488 pre-conjugated rabbit anti-Cleaved Caspase-3 (Cell Signaling, 1:50).

Techniques: Recombinant, Immunoprecipitation, Protease Inhibitor, CRISPR, Construct, Plasmid Preparation, Software