Journal: The Journal of Biological Chemistry

Article Title: Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells

doi: 10.1074/jbc.RA119.010713

Figure Lengend Snippet: Schematic diagram of splice variants of PITX2 and structure of the donor vector used for gene knockin. A, splice variants and isoforms of PITX2. White and red boxes indicate untranslated and coding sequences, respectively. Exons are numbered. B, structure of the donor vector used for establishing the PITX2-based reporter systems. The TAL effector DNA-binding domain (underline) and the location of a silent mutation (C, sense strand; G, antisense strand) are indicated. PITX2, paired-like homeodomain transcription factor 2; CDS, coding sequence; E, exon; TAL, transcription activator–like.

Article Snippet: The RNA was reverse-transcribed into cDNA using the SuperScript III first-strand synthesis system for qRT-PCR (Thermo Fisher Scientific). qRT-PCRs were run using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) and TaqMan minor groove binder probes against GAPDH (Hs99999905_m1), TUBB3 (Hs00801390_s1), SOX10 (Hs00366918_m1), RAX (Hs00429459_m1), PAX6 (Hs00240871_m1), DN-p63 (Hs00978339_m1), CDH1 (Hs01023894_m1), KRT18 (Hs01941416_g1), CRYAA (Hs00166138_m1), PITX2 (Hs00165626_m1), EGFP (Mr04329676_mr), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), p75 (Hs00182120_s1), LMX1B (Hs00158750_m1), TFAP2B (Hs00231468_m1), COL8A2 (Hs00697025_m1), and COL8A1 (Hs00156669_m1) on an ABI Prism 7500 fast sequence detection system (Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Knock-In, Binding Assay, Mutagenesis, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells

doi: 10.1074/jbc.RA119.010713

Figure Lengend Snippet: Protocol for PITX2–EGFP knockin and cloning. A, schematic representation of the protocols for electroporation of TALEN plasmid and PITX2 reporter donor vector into 201B7 hiPSCs grown on drug-resistant MEFs and drug selection. B, PCR analysis of PITX2–EGFP knockin reporter line candidate clones. Odd numbers indicate samples for which 2A peptides were used, and even numbers indicate samples for which IRES2 was used as a polycistronic sequence. Binding sites for primer pairs 1 and 2 are indicated as red and blue arrows, respectively. The amplicon size of primer pair 1 is 598 bp for knockin, whereas amplicon sizes of primer pair 2 are 3677 (3150 when 2A is used) and 1191 bp for knockin and WT, respectively. Arrows indicate the amplicons that prove successful knockin in the candidate clones. C, PCR analysis upon recloning of clone candidate 8. Arrows (clone 8-2) indicate the amplicons that prove successful knockin in the candidate clones of recloning.

Article Snippet: The RNA was reverse-transcribed into cDNA using the SuperScript III first-strand synthesis system for qRT-PCR (Thermo Fisher Scientific). qRT-PCRs were run using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) and TaqMan minor groove binder probes against GAPDH (Hs99999905_m1), TUBB3 (Hs00801390_s1), SOX10 (Hs00366918_m1), RAX (Hs00429459_m1), PAX6 (Hs00240871_m1), DN-p63 (Hs00978339_m1), CDH1 (Hs01023894_m1), KRT18 (Hs01941416_g1), CRYAA (Hs00166138_m1), PITX2 (Hs00165626_m1), EGFP (Mr04329676_mr), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), p75 (Hs00182120_s1), LMX1B (Hs00158750_m1), TFAP2B (Hs00231468_m1), COL8A2 (Hs00697025_m1), and COL8A1 (Hs00156669_m1) on an ABI Prism 7500 fast sequence detection system (Thermo Fisher Scientific).

Techniques: Knock-In, Cloning, Electroporation, Plasmid Preparation, Selection, Clone Assay, Sequencing, Binding Assay, Amplification

Journal: The Journal of Biological Chemistry

Article Title: Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells

doi: 10.1074/jbc.RA119.010713

Figure Lengend Snippet: Confirmation of pluripotency of PITX2–EGFP knockin hiPSC clone 8-2. A, phase-contrast image of 201B7 hiPSCs and PITX2 knockin hiPSC clone 8-2. Scale bars, 200 μm. B, ALP staining of 201B7 hiPSCs and PITX2 knockin hiPSC clone 8-2. The left panels are macro images of staining, and the right panel shows magnifications of the yellow indicated areas. Scale bars, 1000 μm in the left panel and 400 μm in the right panel. C, immunofluorescence staining of pluripotent markers, NANOG, OCT3/4, TRA-1–60, and SSEA-4 in PITX2 knockin hiPSC clone 8-2 cells. Scale bars, 50 μm. D, immunofluorescence staining of SEAMs after 5 weeks for corneal epithelial cells (p63/PAX6), 8 weeks for lens cells (p63/a-crystallin), and 5 weeks for neuroretinal cells (CHX10), and retinal pigment epithelial cell (MITF). Scale bars, 100 μm. E, gene expression patterns in cells that were manually harvested from each SEAM zone induced from PITX2–EGFP knockin hiPSC clone 8-2. Expression levels are presented as dot plots of each value and the means ± standard deviation (n = 4).

Article Snippet: The RNA was reverse-transcribed into cDNA using the SuperScript III first-strand synthesis system for qRT-PCR (Thermo Fisher Scientific). qRT-PCRs were run using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) and TaqMan minor groove binder probes against GAPDH (Hs99999905_m1), TUBB3 (Hs00801390_s1), SOX10 (Hs00366918_m1), RAX (Hs00429459_m1), PAX6 (Hs00240871_m1), DN-p63 (Hs00978339_m1), CDH1 (Hs01023894_m1), KRT18 (Hs01941416_g1), CRYAA (Hs00166138_m1), PITX2 (Hs00165626_m1), EGFP (Mr04329676_mr), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), p75 (Hs00182120_s1), LMX1B (Hs00158750_m1), TFAP2B (Hs00231468_m1), COL8A2 (Hs00697025_m1), and COL8A1 (Hs00156669_m1) on an ABI Prism 7500 fast sequence detection system (Thermo Fisher Scientific).

Techniques: Knock-In, Staining, Immunofluorescence, Gene Expression, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells

doi: 10.1074/jbc.RA119.010713

Figure Lengend Snippet: Correlation between PITX2 and EGFP expression in PITX2–EGFP knockin hiPSC clone 8-2 cells. A, schematic representation of the protocol used for PITX2 induction. B, phase-contrast images of PITX2 knockin hiPSC clone 8-2. Scale bar, 200 μm. C, phase-contrast and fluorescence images of aggregated PITX2 knockin hiPSC clone 8-2 that express EGFP fluorescence. Scale bar, 100 μm in the left panel and 50 μm in the right panel. D, Western blotting analysis of PITX2 (61 kDa), EGFP (27 kDa), and GAPDH (37 kDa) in PITX2 knockin hiPSC clone 8-2. E, image of immunofluorescence staining of PITX2 knockin hiPSC clone 8-2 cells that express PITX2 (red) and EGFP fluorescence (green). Scale bar, 50 μm. F, FACS analysis of EGFP fluorescence intensity in 201B7 hiPSCs and PITX2 knockin hiPSC clone 8-2. The cells were expanded in PE (y axis) and FITC (x axis). The cells were selected using polygon gates; cells in the blue polygon are EGFP-negative, and those in the purple polygon are EGFP-positive. G, mRNA expressions of positive and negative markers of POM cell as determined by qRT-PCR in EGFP-negative and -positive cells after cell sorting. Expression levels are presented as dot plots of each value and the means ± standard deviation (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. N, negative cells; P, positive cells.

Article Snippet: The RNA was reverse-transcribed into cDNA using the SuperScript III first-strand synthesis system for qRT-PCR (Thermo Fisher Scientific). qRT-PCRs were run using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) and TaqMan minor groove binder probes against GAPDH (Hs99999905_m1), TUBB3 (Hs00801390_s1), SOX10 (Hs00366918_m1), RAX (Hs00429459_m1), PAX6 (Hs00240871_m1), DN-p63 (Hs00978339_m1), CDH1 (Hs01023894_m1), KRT18 (Hs01941416_g1), CRYAA (Hs00166138_m1), PITX2 (Hs00165626_m1), EGFP (Mr04329676_mr), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), p75 (Hs00182120_s1), LMX1B (Hs00158750_m1), TFAP2B (Hs00231468_m1), COL8A2 (Hs00697025_m1), and COL8A1 (Hs00156669_m1) on an ABI Prism 7500 fast sequence detection system (Thermo Fisher Scientific).

Techniques: Expressing, Knock-In, Fluorescence, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, FACS, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells

doi: 10.1074/jbc.RA119.010713

Figure Lengend Snippet: POM cells expressed in SEAMs in PITX2–EGFP knockin hiPSC clone 8-2 cells. A, schematic representation of the protocol used for SEAM induction. B, phase-contrast images of PITX2 knockin hiPSC clone 8-2. Scale bar, 200 μm. C, phase-contrast and fluorescence images of aggregated PITX2 knockin hiPSC clone 8-2 that express EGFP. Scale bars, 100 μm in the left panel and 50 μm in the right panel. D, FACS analysis of EGFP fluorescence intensity in 201B7 hiPSCs and PITX2 knockin hiPSC clone 8-2. The cells were expanded in PE (y axis) and FITC (x axis). The cells in the blue polygon are EGFP-negative, and those in the purple polygon are EGFP-positive. E, mRNA expressions of positive and negative markers of POM cell as determined by qRT-PCR in EGFP-negative and -positive cells after cell sorting. Expression levels are presented as dot plots of each value and the means ± standard deviation (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. N, negative cells; P, positive cells. F, phase-contrast image of POM cells after 2 days of culture. G, immunofluorescence images of nuclei (blue), PITX2 (red), and FOXC1 (gray) in POM cells after 2 days of culture.

Article Snippet: The RNA was reverse-transcribed into cDNA using the SuperScript III first-strand synthesis system for qRT-PCR (Thermo Fisher Scientific). qRT-PCRs were run using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) and TaqMan minor groove binder probes against GAPDH (Hs99999905_m1), TUBB3 (Hs00801390_s1), SOX10 (Hs00366918_m1), RAX (Hs00429459_m1), PAX6 (Hs00240871_m1), DN-p63 (Hs00978339_m1), CDH1 (Hs01023894_m1), KRT18 (Hs01941416_g1), CRYAA (Hs00166138_m1), PITX2 (Hs00165626_m1), EGFP (Mr04329676_mr), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), p75 (Hs00182120_s1), LMX1B (Hs00158750_m1), TFAP2B (Hs00231468_m1), COL8A2 (Hs00697025_m1), and COL8A1 (Hs00156669_m1) on an ABI Prism 7500 fast sequence detection system (Thermo Fisher Scientific).

Techniques: Knock-In, Fluorescence, Quantitative RT-PCR, FACS, Expressing, Standard Deviation, Immunofluorescence

Journal: bioRxiv

Article Title: Mmp14-controlled extracellular matrix homeostasis is required for circadian rhythm

doi: 10.1101/2022.04.01.486699

Figure Lengend Snippet: A , Real-time PCR detection of Pitx2 mRNA levels in WT and Mmp14 CKO tail tendons 5 weeks post tamoxifen treatment, at ZT3 (09:00 hr) and ZT15 (21:00 hr). B , Real-time PCR of PLOD2 mRNA expression in WT and Mmp14 CKO tail tendons (n=3 animals per time point) 5 weeks post tamoxifen treatment, at ZT3 (09:00 hr) and ZT15 (21:00 hr). C , Per2::Luc iTTFs stably transduced with control or Pitx2-GFP lentivirus, demonstrating enhanced expression of Pitx2 and a concomitant increase in Plod2 mRNA expression D , (n=3 and n=5 respectively). E , Live cell Airyscan imaging of Per2::Luc iTTFs and MMP14 CRISPR-Cas9 KO ITTFs stably transduced with Pitx2-GFP lentivirus. Cells were transiently transduced with mCherry tagged MMP14 carrying a silent mutation in the CRISPR-Cas9 PAM site. Pitx2-GFP was readily detected within the nucleus but also with punctate localisation within the cell body in control cell, these correlated with regions also positive for MT1-MMP. Nuclear Pitx2-GFP signals were stronger within Mmp14 KO cells and also displayed reduced localisation within the cell body. Re-expression of Mmp14 within knockout cells reduced nuclear Pitx-GFP signals. F-I , Real-time PCR demonstrating expression of Mmp14 in Pitx2-GFP expressing iTTFs, which resulted in reduced Pitx2 and Plod2 mRNA expression, but enhanced Col1a1 mRNA (n=3).

Article Snippet: Pitx2GFP expressing cells were cloned into pLenti PGK Neo DEST (Addgene plasmid # 19067 ref ), EGFP was cloned at the C terminus of the coding sequence of Pitx2 isoform 1 (Origene; MR227617).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Transduction, Imaging, CRISPR, Mutagenesis, Knock-Out

Journal: bioRxiv

Article Title: Mmp14-controlled extracellular matrix homeostasis is required for circadian rhythm

doi: 10.1101/2022.04.01.486699

Figure Lengend Snippet: A , Bioluminescence recordings from Per2::Luc iTTFs and the same cells stably overexpressing Pitx2 and Pitx2-GFP after synchronisation with dexamethasone. B , bioluminescence recordings from Per2::Luc iTTFs (n=3) and Pitx2 overexpressing fibroblasts that have been transduced with a lentivirus expressing Pitx2.

Article Snippet: Pitx2GFP expressing cells were cloned into pLenti PGK Neo DEST (Addgene plasmid # 19067 ref ), EGFP was cloned at the C terminus of the coding sequence of Pitx2 isoform 1 (Origene; MR227617).

Techniques: Stable Transfection, Transduction, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Expression of PITX2 homeodomain transcription factor during rat gonadal development in a sexually dimorphic manner.

doi: 10.1159/000325218

Figure Lengend Snippet: Fig. 1. Analysis of variation in rat Pitx2/PITX2 isoforms, and epitope mapping followed by western validation of the PITX2 specific antibody in rat, human and mice. (A) CLUSTAL W generated Multiple Sequence Alignment (CLUSTAL W MSA) shows the variation in the m-RNA CDS region of Pitx2a, -b and -c isoforms and their homology with variant wise nomenclature Pitx2 varaint1 and variant2 of rat. The available NCBI GenBank accession number and gene nomenclature are shown with each sequence. The rat Pitx2c CDS sequence partially available in NCBI GenBank and termed here as rPitx2c-per (r, rat). (B) CLUSTAL W MSA shows the variation in the N-terminal region of rat PITX2A, -B and -C isoforms and their homology with PITX2 varaint1 and variant2 of rat. The available NCBI Protein Bank accession number and nomenclature are shown with each sequence. (r, rat). (C) CLUSTAL W MSA of rat PITX2A, -B and -C protein epitope alignment with other orthologues from human and mice showing the conserved nature of 15 amino acid long epitope sequence. The epitope is indicated by box containing the colour case letters from where the antibody was raised. The NCBI Protein Bank accession number and nomenclature of isoforms are shown with each sequence. (r, rat; m, mice; h, human). (D) Immunoblot analysis of PITX2 isoforms expressed in SKOV-3 ovarian cell line detects all spliced variants of PITX2. (Are PITX2A, -B1, -B2, -CD and -CE, lane 1 and 2). (E) Immunoblot analysis of rat embryonic ovary and testis protein shows expression of four PITX2 isoforms which correspond to PITX2B1, -B2, -CD and -CE (lane 1;

Article Snippet: The Taqman gene expression assay primers and probes specific to rat Pitx2b and -c and -Actin (Assay ID- Rn00571000_m1, Rn01521836_m1 and part no4352931E respectively) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Western Blot, Biomarker Discovery, Generated, Sequencing, Variant Assay, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Expression of PITX2 homeodomain transcription factor during rat gonadal development in a sexually dimorphic manner.

doi: 10.1159/000325218

Figure Lengend Snippet: Fig. 2. Comparative gene expression profile of Pitx2b and c in rat gonads at embryonic and postnatal stages. Total RNAs from ovary (O) and testis (T) were isolated at embryonic days E14.5, 16.5, 18.5, and 20.5, and at postnatal days, D10 and 30 followed by Taqman Q-PCR using rat Pitx2b and Pitx2c gene-specific primers and probes. Fig 2(A) and (B) show the Q-PCR results of these two isoform with ovarian and testicular RNAs respectively. The data are represented by relative gene expression in fold change calculated by 2-''CT method. The CT -values of Pitx2 gene are normalized from the CT -value of the housekeeping gene E-Actin. The gene expression level of Pitx2c at E14.5 ovary/testis was considered as 1. The embryonic and the postnatal developmental days of ovary and testis have been mentioned. The experiments were performed three times and the mean ± SE value have been shown, *, P<0.05.

Article Snippet: The Taqman gene expression assay primers and probes specific to rat Pitx2b and -c and -Actin (Assay ID- Rn00571000_m1, Rn01521836_m1 and part no4352931E respectively) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Gene Expression, Isolation

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Expression of PITX2 homeodomain transcription factor during rat gonadal development in a sexually dimorphic manner.

doi: 10.1159/000325218

Figure Lengend Snippet: Fig. 3. Comparative PITX2 protein isoform expression profile in embryonic and postnatal rat gonads. Whole tissue proteins from the embryonic (E14.5, 16.5, 18.5, 20.5) and postnatal gonads (D10, 30) were isolated as mentioned in methods section. The protein samples were subjected to 12% SDS-PAGE followed by western immuno-detection using PITX2 specific antibody. Fig. 3A shows that the PITX2 isoforms that express throughout the embryonic gestational days at E14.5, 16.5, 18.5 and 20.5 rat gonads are predominantly PITX2B2 and -CE whereas there with relatively lower level expression are PITX2B1 and CD (Fig. 3A bands above -B2). Fig. 3B shows that PITX2B2 and -CE are the isoforms expressed at postnatal D10 and 30 gonads. The pixel densities of the individual isoforms were measured using ImageJ software (NIH, USA) and are shown below the specific lanes; respective isoforms are indicated with arrow. Below each bar diagram, the day and the tissue types have been mentioned. The experiments were performed three times and the mean ± SE value have been shown, *, P<0.05. ‘O’-Ovary and ‘T’-Testis.

Article Snippet: The Taqman gene expression assay primers and probes specific to rat Pitx2b and -c and -Actin (Assay ID- Rn00571000_m1, Rn01521836_m1 and part no4352931E respectively) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Expressing, Isolation, SDS Page, Western Blot, Software

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Expression of PITX2 homeodomain transcription factor during rat gonadal development in a sexually dimorphic manner.

doi: 10.1159/000325218

Figure Lengend Snippet: Fig. 6. Localization of PITX2 at postnatal ovary and testis. The ovary (Fig. 6A) and testis (Fig. 6B) were isolated at D10 and D30 followed by paraffin block preparation, sectioning and subsequent IHC studies. In Fig. 6A, the right upper and lower panels show the localization of PITX2 in the differentiating ovarian follicles at D10 and 30 respectively. At D30, PITX2 intra-follicular localization was observed especially in granulosa (arrows) and theca cells (arrow heads). In Fig. 6B the right upper and lower panels show the localization of PITX2 in the spermatogonia (arrow head) and primary spermatocytes (arrows) of seminiferous tubules at D10 and D30 testis respectively. Consecutive sections of each sample were stained in hematoxylin and eosin and are shown in the left panel. For the sake of brevity, images of the selected days are furnished to highlight the pattern of developmental expression. The Negative Control images represent the staining in presence of secondary antibody but without PITX2 antibody. Scale bars: red, 100 μm; yellow, 50 μm; black, 15 μm. In Fig. 6A, arrows in upper panel shows growing follicles, arrows in lower panel -granulosa cells, arrowhead -theca cells. In Fig. 6B, arrow shows spermatocytes, arrowhead -spermatogonia, asterisks -intertubular blood vessels.

Article Snippet: The Taqman gene expression assay primers and probes specific to rat Pitx2b and -c and -Actin (Assay ID- Rn00571000_m1, Rn01521836_m1 and part no4352931E respectively) were purchased from Applied Biosystems (Foster City, CA, USA).

Techniques: Isolation, Blocking Assay, Staining, Expressing, Negative Control