cu1633  (Developmental Studies Hybridoma Bank)

Journal: iScience

Article Title: The Iroquois ( Iro/Irx ) homeobox genes are conserved Hox targets involved in motor neuron development

doi: 10.1016/j.isci.2025.112210

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Lim3/Lhx3 , Developmental Studies Hybridoma Bank , Cat#67.4E12; RRID: AB_2135805.

Techniques: Virus, Recombinant, Blocking Assay, Electron Microscopy, Saline, Software, Transgenic Assay

Journal: iScience

Article Title: The Iroquois ( Iro/Irx ) homeobox genes are conserved Hox targets involved in motor neuron development

doi: 10.1016/j.isci.2025.112210

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Lim3/Lhx3 , Developmental Studies Hybridoma Bank , Cat67.4E12; RRID: AB_2135805.

Techniques: Virus, Recombinant, Blocking Assay, Electron Microscopy, Saline, Software, Transgenic Assay

Journal: iScience

Article Title: The Iroquois ( Iro/Irx ) homeobox genes are conserved Hox targets involved in motor neuron development

doi: 10.1016/j.isci.2025.112210

Figure Lengend Snippet: Expression of Irx1, Irx2, Irx3, Irx5, and Irx6 in specific motor columns (A) Brachial spinal domain (C4-T1) in an e12.5 mouse. Cross-section: LMC and MMC neurons at the ventral region of the spinal cord. (B) Irx1 FISH coupled with Lhx3 antibody staining shows Irx1 expression (red) is detected in Lhx3-expressing MNs (green) of the MMC. (C) Schematic of Irx1 expression. (D and E) Irx2 FISH combined with immunostaining for Foxp1 (D) or Lhx3 (E) reveals Irx2 expression (red, also converted to white for better contrast) is detected in LMC (D) and MMC (E) neurons. (F) Schematic of Irx2 expression. (G) Double immunostaining for Irx3 and Lhx3 shows Irx3 (red) is expressed in a small population of Lhx3-positive MNs (green). (H) Irx3 antibody staining combined with Pea3 RNA FISH reveals almost no co-localization of Irx3 protein (red) to Pea3 -expressing MNs (green) of the ventral LMC but expression in a small population of dorsal LMC MNs. (I) Schematic of Irx3 expression. (J) Double immunofluorescence reveals Irx5 expression (red) in Hb9-positive cells (green). (K) Two-color RNA FISH shows Irx5 expression (red) is detected in Pea3 -expressing MNs of the ventral LMC. (L) Schematic of Irx5 expression. (M) Irx6 RNA (red) colocalizes with Foxp1 protein (green) in the ventral LMC. (N) Two-color RNA FISH reveals Irx6 expression (red) in Pea3 -expressing MNs. (O) Schematic of Irx6 expression. B-O: e12.5 spinal cord, N = 4. (P-Q) Dot plots show Irx1 , Irx2 , Irx3 , Irx5 , and Irx6 transcripts in MN subclusters/pools previously identified in the e13.5 mouse brachial spinal cord. scRNA-Seq data analyzed from. (P) LMC subclusters/pools are defined by the combinatorial expression of various TFs. Known MN pool markers include: Etv1/Er81, Etv4/Pea3, Pou 3 f1/Scip . (Q) Three MMC subclusters/pools previously defined by Bcl11b, Satb2 , or Nr2f expression. rm: rostral medial; rl: rostral lateral; cl: caudal lateral; cm: caudal medial. Size of dots indicates the percentage of cells expressing each Irx gene. Color code indicates average expression levels. Scale bar: 50 μm. See also Figures S1 and .

Article Snippet: Then, the anti-DIG antibody conjugated with peroxidase (Roche) and primary antibody against Foxp1 (rabbit anti-Foxp1, Dr. Jeremy Dasen), Irx3 (rabbit anti-Irx3, Abcam), or Lhx3 (mouse anti-Lim3, Developmental Studies Hybridoma Bank) were applied overnight (4°C) to the sections.

Techniques: Expressing, Staining, Immunostaining, Double Immunostaining, Immunofluorescence

Journal: iScience

Article Title: The Iroquois ( Iro/Irx ) homeobox genes are conserved Hox targets involved in motor neuron development

doi: 10.1016/j.isci.2025.112210

Figure Lengend Snippet: Generation and characterization of Irx5 mutant mice (A) A gRNA targets exon 1 of Irx5 . Representative chromatograms of genotyped mice illustrate the 5 bp (CTCGC) deletion in Irx5 Δ 5bp/ Δ 5bp . (B) Wild-type and truncated Irx5 proteins produced in the Irx5 Δ 5bp mouse. Both HD and Iro domains are removed in the Irx5 Δ 5bp protein. (C) Immunohistochemistry shows strong reduction in Irx5 expression in Irx5 Δ 5bp/ Δ 5bp spinal cords at e12.5. (D) Double immunohistochemistry for Hb9 and Lhx3 in control and Irx5 Δ 5bp/ Δ 5bp mice at e12.5. (E) Double immunohistochemistry for Foxp1 and Pea3 in control and Irx5 Δ 5bp/ Δ 5bp mice at e12.5. (F–H) Quantification of Hb9+, Foxp1+, or Pea3+ MNs in Irx5 Δ 5bp/ Δ 5bp mice at e12.5. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, N. S.: Not significant. C-H: N = 2–3 for Irx5 + / + , N = 2 for Irx5 Δ 5bp/ Δ 5bp . Analyses performed using unpaired t -test (two-tailed). Data are shown as mean ± SEM. (I) Immunostaining shows ChAT expression in control and Irx5 Δ 5bp/ Δ 5bp spinal cords at p10. (J and K) Quantification of ChAT+ (J) or ChAT+ and Isl1/2+ double-positive cells (K) in control and Irx5 Δ 5bp/ Δ 5bp spinal cords at p10. N = 3 (L-M) MN axons labeled with Hb9GFP in control and Irx5 Δ 5bp/ Δ 5bp mice at e12.5. Projections to forelimb (L) and cutaneous maximus (M) muscle. I-J: N = 3 for Irx5 + / + , N = 2 for Irx5 Δ 5bp/ Δ 5bp . Scale bar: 50 μm.

Article Snippet: Then, the anti-DIG antibody conjugated with peroxidase (Roche) and primary antibody against Foxp1 (rabbit anti-Foxp1, Dr. Jeremy Dasen), Irx3 (rabbit anti-Irx3, Abcam), or Lhx3 (mouse anti-Lim3, Developmental Studies Hybridoma Bank) were applied overnight (4°C) to the sections.

Techniques: Mutagenesis, Produced, Immunohistochemistry, Expressing, Control, Two Tailed Test, Immunostaining, Labeling

Journal: iScience

Article Title: The Iroquois ( Iro/Irx ) homeobox genes are conserved Hox targets involved in motor neuron development

doi: 10.1016/j.isci.2025.112210

Figure Lengend Snippet:

Article Snippet: Then, the anti-DIG antibody conjugated with peroxidase (Roche) and primary antibody against Foxp1 (rabbit anti-Foxp1, Dr. Jeremy Dasen), Irx3 (rabbit anti-Irx3, Abcam), or Lhx3 (mouse anti-Lim3, Developmental Studies Hybridoma Bank) were applied overnight (4°C) to the sections.

Techniques: Virus, Recombinant, Blocking Assay, Electron Microscopy, Saline, Software, Transgenic Assay

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet: ( A ) Culture protocol and outline to generate pituitary organoids in three-dimensional (3D) culture from human induced pluripotent stem cell (hiPSC). Organoids were collected at days ( d ) 0, 6, 18, 27, 48, 75, and 105 during differentiation to analyze. ( B ) Brightfield microscopy views of WT organoid examples at different time points throughout differentiation. Scale bars are indicated in each image. ( C ) Number of replicates per time point and per genotype in experiments depicted in the following graphs. Asterisks indicate that 7–8 organoids were grouped for each sample. For other points, each sample consists of a single organoid. ( D–H ) Relative quantification (RQ) mRNA expression analysis for key markers of pituitary organoids during differentiation: WT (in black line) and TBX19 KI organoids (in red line). Relative quantification of each target gene was obtained by the 2 -ΔΔCt method from qRT-PCR results (see Methods). Data show means ± standard error of the mean (SEM, Mann-Whitney t-test [unpaired, two-tailed, nonparametric]). p<0.05 (*), p<0.01 (**). ( D ) Relative quantification of HESX1 expression, the earliest pituitary placode marker assessed. The expression of HESX1 is significantly downregulated in TBX19 KI organoids vs. WT at d18 and d27. ( E ) Relative quantification expression of PITX1 , a pituitary progenitor marker. PITX1 was significantly downregulated in TBX19 KI organoids by d48 and d75. ( F ) Relative quantification expression of LHX3 , a pituitary progenitor marker. LHX3 was significantly lower in TBX19 KI organoids as compared to WT from d48 onwards. ( G ) Relative quantification expression of TBX19 , a critical transcriptional determinant for corticotroph differentiation. TBX19 expression is higher in TBX19 KI organoids at d105. ( H ) Relative quantification expression of pro-opiomelanocortin ( POMC) , a corticotroph marker. POMC was significantly downregulated in TBX19 KI organoids from d48 onwards.

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques: Microscopy, Quantitative Proteomics, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Two Tailed Test, Marker

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet: ( A–C ) Induction of two tissues hypothalamus-pituitary in organoid on day 48. ( A ) Oral ectoderm-like tissue expressed pituitary progenitor markers (LHX3). ( B ) Hypothalamus-like tissue expressed hypothalamic progenitor markers (NKX2.1). ( C ) Oral ectoderm-like tissue expressed PITX1 and E-cadherin. ( D ) Corticotroph cells expressed TBX19 in the nucleus and adrenocorticotropic hormone (ACTH) in the cytoplasm in organoid on day 105. Scale bars: 50 µm.

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques:

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet: ( A, B ) Immunostaining of LHX3 and CDH1 (E-cadherin) expression in epithelial cells, typical of Rathke’s pouch ectoderm in early pituitary primordia, was reduced in TBX19 KI organoid vs wild-type (WT) on day 48 (n=10 organoids for each group). Scale bars: 10 μm. ( C, D ) Immunostaining showed that adrenocorticotropic hormone (ACTH) and TBX19 expressions were reduced in TBX19 KI organoid vs WT on day 105 (n=10 organoids for each group). Scale bars: 10 μm.

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques: Immunostaining, Expressing

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet: ( A ) Number of replicates per time point and per genotype in experiments depicted in the following graphs. Asterisks indicate that 7–8 organoids were grouped for each sample. For other point, each sample consists of a single organoid. ( B–F ) Relative quantification (RQ) mRNA expression analysis for key markers of pituitary organoids during differentiation: WT (black line and dots) and NFKB2 KI organoids (red line and dots). Data show means ± standard error of the mean (SEM);. Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p<0.05 (*), p<0.01 (**). ( B ) Relative quantification expression of HESX1 , the earliest pituitary placode marker assessed. The expression of HESX1 was upregulated in NFKB2 KI organoids vs. WT between d27 and d75. ( C ) Pituitary progenitor marker PITX1 was significantly downregulated in NFKB2 KI organoids at d48 and d75. ( D ) Pituitary progenitor marker LHX3 was significantly lower in NFKB2 KI organoids as compared to WT from d48 onwards. ( E ) Relative quantification expression of TBX19 , a corticotroph marker. TBX19 was significantly increased in NFKB2 KI organoids by d105. ( F ) Relative quantification expression of pro-opiomelanocortin ( POMC) , a corticotroph marker. POMC was significantly downregulated in NFKB2 KI organoids from d75. ( G ) Volume of organoids (mm 3 ) on d105, calculated using Imaris software (see in methods). There was no significant difference in volume between WT and NFKB2 KI organoids (p=0.6126). Data show means ± SEM; n=7 in the WT group, n=8 in the mutant group. Mann-Whitney test (unpaired, two-tailed).

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques: Quantitative Proteomics, Expressing, MANN-WHITNEY, Two Tailed Test, Marker, Software, Mutagenesis

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet: ( A, B ) Immunostaining for LHX3 (green) and p100/p52 (red) in the early pituitary-type epithelium was reduced in NFKB2 KI organoids vs wild-type (WT) on day 48 (n=10 organoids for each group). Stronger expression of p100/p52 was observed in pituitary progenitors, but expression in the hypothalamic part of the organoid cannot be excluded. Scale bar: 10 μm ( C, D ) by day 105, although nuclear TBX19 was detectable in both mutants, these cells failed to co-express ACTH in NFKB2 KI organoids (n=10 organoids for each group). Scale bars: 10 μm.

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques: Immunostaining, Expressing

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet: RNA-seq expression data for a list of 144 genes known from the literature to have a functional influence on pituitary-hypothalamic development. Differentially expressed genes (padj <0.05) in nuclear factor kappa-B subunit 2 ( NFKB2) KI organoids are highlighted in green when upregulated and in orange when downregulated. FC; Fold change.

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques: Expressing, Functional Assay

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet: ( A ) RT-qPCR experiments at d48 show that HESX1 expression is upregulated NFKB2 KI organoids, compared to wild-type (WT) and TBX19 KI, whereas PITX1 and LHX3 are downregulated in both mutants. ( B ) BMP4 expression is increased whereas, and FGF8 and FGF10 are decreased in NFKB2 mutants, but unchanged in TBX19 KI organoids. Data show means ± SEM ; n=17, 12, and 11 for WT, TBX19 KI, and NFKB2 KI, respectively. Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p<0.05 (*), p<0.01 (**), p<0.005 (***),.

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques: Quantitative RT-PCR, Expressing, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation

doi: 10.7554/eLife.90875

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human LHX3 Mouse monoclonal , DSHB , 67.4E12 RRID: AB_2135805 , (1/500).

Techniques: Cell Culture, Knock-Out, Recombinant, Sequencing, CRISPR