Journal: Scientific Reports

Article Title: The catalytic activity and secretion of zebrafish RNases are essential for their in vivo function in motor neurons and vasculature

doi: 10.1038/s41598-018-37140-2

Figure Lengend Snippet: Antibodies used in this study and dilutions.

Article Snippet: 40-2D6 (Isl1/2) , Mouse, IgG1 , 1:5 , DSHB.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

doi: 10.1523/JNEUROSCI.3046-14.2015

Figure Lengend Snippet: Primary antibodies

Article Snippet: Blocking was performed in PBS containing 1% BSA supplemented with 0.3% Triton for permeabilization for 30 min to 1 h. Cells were incubated in primary antibodies diluted in blocking buffer (without Triton) overnight at 4°C, followed by several washes with PBS, and then incubation in Alexa Fluor-labeled secondary antibodies (Invitrogen) diluted 1:400 in blocking buffer for 30 min to 1 h. Please refer to the antibody list in for details on sources and concentrations. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Species, isotype Company Dilution GFP Chicken Abcam 1:1000 HB9 Mouse, IgG1 DSHB 1:100 NKX6.1 Mouse, IgG1 DSHB 1:100 ISL1/2 Mouse, IgG2b DSHB 1:100 LIM3 Mouse, IgG1 DSHB 1:100 CHAT Goat Chemicon 1:100 TUJ1 Mouse or rabbit Covance 1:500 MAP2 Mouse Sigma-Aldrich 1:200 PAX6 Rabbit Covance 1:75 SMI32 Mouse Millipore 1:1000 OLIG2 Rabbit Millipore 1:200 FOXA2 Goat Santa Cruz Biotechnology 1:100 GLI3 Rabbit Wang et al., 2000 1:100 Open in a separate window DSHB, Developmental Studies Hybridoma Bank.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

doi: 10.1523/JNEUROSCI.3046-14.2015

Figure Lengend Snippet: Efficient derivation of MNs from hESCs upon early exposure to patterning cues. Exposure to patterning factors at a very early neural stage (A) during dSMADi for at least 2 weeks (B) efficiently specifies MNs as assessed by percentage of GFP+ cells using an HB9::GFP BAC hESC line. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05 versus RA initiated at day 1 (A) and LSB (B), Dunnett test. C, SHH exposure in place of or in addition to Pur does not improve yields. Data are shown as mean ± SEM; n = 2 independent experiments versus RA + Pur, Dunnett test. D, Acquisition of GFP occurs rapidly, with cultures expressing up to 40% GFP+ after 2 weeks of patterning. E, Representative FACS plot for GFP at day 16 for LSB and RA + Pur. F, Live GFP expression in LSB and RA + Pur cultures at day 16. Scale bar, 10 μm. G, Isolated GFP+ cells at day 10 already coexpress ISL1 (blue) and TUJ1 (red) by immunocytochemistry. Scale bar, 10 μm. H, Immunocytochemistry at day 7 for PAX6 (red) and NKX6.1 (green) demonstrates presence of MN progenitors within 1 week of differentiation. Scale bars, 100 μm. I, Immunocytochemistry at day 16 for OLIG2, HB9, ISL1, and LIM3 verifies MN identity. Scale bars, 100 μm. J, K, After 3 weeks of differentiation, MNs exhibit more mature features including neuronal morphology as shown by immunocytochemistry for HB9 (red), ISL1 (blue), and GFP (green) of GFP+ isolated cells at day 19 and by expression of ChAT (green) and ISL1 (red) at day 26. Scale bars, 10 μm. L, Quantification of markers presented in I. M, N, Evaluation of patterning factors individually reveals striking ability of RA to induce MN markers HB9 and ISL1 efficiently in the presence and absence of SHH activation (Pur) as assessed by qRT-PCR.

Article Snippet: Blocking was performed in PBS containing 1% BSA supplemented with 0.3% Triton for permeabilization for 30 min to 1 h. Cells were incubated in primary antibodies diluted in blocking buffer (without Triton) overnight at 4°C, followed by several washes with PBS, and then incubation in Alexa Fluor-labeled secondary antibodies (Invitrogen) diluted 1:400 in blocking buffer for 30 min to 1 h. Please refer to the antibody list in for details on sources and concentrations. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Species, isotype Company Dilution GFP Chicken Abcam 1:1000 HB9 Mouse, IgG1 DSHB 1:100 NKX6.1 Mouse, IgG1 DSHB 1:100 ISL1/2 Mouse, IgG2b DSHB 1:100 LIM3 Mouse, IgG1 DSHB 1:100 CHAT Goat Chemicon 1:100 TUJ1 Mouse or rabbit Covance 1:500 MAP2 Mouse Sigma-Aldrich 1:200 PAX6 Rabbit Covance 1:75 SMI32 Mouse Millipore 1:1000 OLIG2 Rabbit Millipore 1:200 FOXA2 Goat Santa Cruz Biotechnology 1:100 GLI3 Rabbit Wang et al., 2000 1:100 Open in a separate window DSHB, Developmental Studies Hybridoma Bank.

Techniques: Expressing, Isolation, Immunocytochemistry, Activation Assay, Quantitative RT-PCR

Journal: The Journal of Neuroscience

Article Title: Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

doi: 10.1523/JNEUROSCI.3046-14.2015

Figure Lengend Snippet: RA induces MNs from hESCs and iPSCs in the absence of extrinsic SHH activation. A, Early exposure to RA efficiently derives MNs from hPSCs with comparable yields and timing in the presence and absence of Pur. B, Patterning with RA achieves highest MN yields when initiated at a very early neural stage (day 1). Data are shown as mean ± SEM; n = 3 independent experiments; ***p < 0.001; **p < 0.01; *p < 0.05 versus RA initiated at day 1, Dunnett test. C, Live GFP expression in RA-patterned culture at day 16. Scale bar, 10 μm. Immunocytochemistry at day 7 for PAX6 (D), at day 16 for HB9, ISL1, and LIM3 (E), and at day 21 for NKX6.1 (green) and OLIG2 (red; F) confirms MN identity of RA-derived cultures. G, Quantification of markers presented in E and F. Immunocytochemistry at day 23 for HB9, ISL1, and TUJ1 (H) shows MNs at a more mature stage. I–K, Immunocytochemistry at day 16 for ISL1 (red) and HB9 (green; left), NKX6.1 (green) and TUJ1 (red; middle), and at day 23 for SMI32 (green) and ISL1 (red; right) for MNs derived from control (11A and 18B) and ALS patient-derived (27B) iPSCs via patterning with RA (J) or RA + Pur (K). I, Quantification of markers in J and K demonstrating similar, efficient propensity to generate MNs across multiple lines regardless of disease status. Scale bars, 100 μm unless labeled otherwise.

Article Snippet: Blocking was performed in PBS containing 1% BSA supplemented with 0.3% Triton for permeabilization for 30 min to 1 h. Cells were incubated in primary antibodies diluted in blocking buffer (without Triton) overnight at 4°C, followed by several washes with PBS, and then incubation in Alexa Fluor-labeled secondary antibodies (Invitrogen) diluted 1:400 in blocking buffer for 30 min to 1 h. Please refer to the antibody list in for details on sources and concentrations. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Species, isotype Company Dilution GFP Chicken Abcam 1:1000 HB9 Mouse, IgG1 DSHB 1:100 NKX6.1 Mouse, IgG1 DSHB 1:100 ISL1/2 Mouse, IgG2b DSHB 1:100 LIM3 Mouse, IgG1 DSHB 1:100 CHAT Goat Chemicon 1:100 TUJ1 Mouse or rabbit Covance 1:500 MAP2 Mouse Sigma-Aldrich 1:200 PAX6 Rabbit Covance 1:75 SMI32 Mouse Millipore 1:1000 OLIG2 Rabbit Millipore 1:200 FOXA2 Goat Santa Cruz Biotechnology 1:100 GLI3 Rabbit Wang et al., 2000 1:100 Open in a separate window DSHB, Developmental Studies Hybridoma Bank.

Techniques: Activation Assay, Expressing, Immunocytochemistry, Derivative Assay, Labeling

Journal: The Journal of Neuroscience

Article Title: Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

doi: 10.1523/JNEUROSCI.3046-14.2015

Figure Lengend Snippet: Functional characterization of MNs patterned with RA alone versus RA + Pur. A, Differential interference contrast photomicrograph of a representative patch-clamped MN at 38 d of differentiation. Scale bar, 20 μm. B, Graphs showing the comparison of input resistance and resting membrane potential (RMP) of RA-alone- versus RA + Pur-induced MNs. *p < 0.05. C, Spontaneous APs (at the indicated membrane potential) were readily observed in an RA + Pur-induced MN. D, In a different RA + Pur-derived neuron than in C, a representative voltage trace demonstrating sustained, tonic AP firing in response to a 30 pA depolarizing current step (current protocol shown below). E, Graph showing relation between firing frequency and injected current pulse. Data are shown as mean ± SEM of all the data points from RA (n = 15) and RA + Pur (n = 9) until 25 pA, after which the number of data points are indicated for each current step. F, AP firing was blocked by the Na+ channel blocker tetrodotoxin (TTX) in an RA-only-derived neuron. G, Immunocytochemistry for ISL1 and SMI32 (left) or DAPI (right) in biocytin-filled cells confirms MN identity of recorded cells corresponding to D and F. Scale bars, 10 μm. H–L, Both RA- and RA + Pur-derived MNs exhibit an excitatory calcium response after glutamate exposure. Representative ratiometric images of identical MNs in the absence and presence of glutamate (100 μm) patterned with RA (H) or RA + Pur (I). Calcium traces in response to glutamate activation (J) and inhibition of this response by application of glutamate receptor antagonists AP5 (50 μm) and DNQX (10 μm; K) in RA-derived MNs. L, Quantification of activation in response to glutamate exposure in the presence and absence of AP5 and/or DNQX (RA n = 16, RA + Pur n = 16). There was no significant difference in activation levels between MNs derived with RA versus those derived with RA + Pur. Data are shown as mean ± SEM; ****p < 0.0001 versus GLU, Dunnett test. M, Quantification of spontaneous contractions observed upon coculture of RA-derived MNs and C2C12 myoblasts that are suppressed by application of the acetylcholine receptor antagonist vecuronium (2 μm).

Article Snippet: Blocking was performed in PBS containing 1% BSA supplemented with 0.3% Triton for permeabilization for 30 min to 1 h. Cells were incubated in primary antibodies diluted in blocking buffer (without Triton) overnight at 4°C, followed by several washes with PBS, and then incubation in Alexa Fluor-labeled secondary antibodies (Invitrogen) diluted 1:400 in blocking buffer for 30 min to 1 h. Please refer to the antibody list in for details on sources and concentrations. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Species, isotype Company Dilution GFP Chicken Abcam 1:1000 HB9 Mouse, IgG1 DSHB 1:100 NKX6.1 Mouse, IgG1 DSHB 1:100 ISL1/2 Mouse, IgG2b DSHB 1:100 LIM3 Mouse, IgG1 DSHB 1:100 CHAT Goat Chemicon 1:100 TUJ1 Mouse or rabbit Covance 1:500 MAP2 Mouse Sigma-Aldrich 1:200 PAX6 Rabbit Covance 1:75 SMI32 Mouse Millipore 1:1000 OLIG2 Rabbit Millipore 1:200 FOXA2 Goat Santa Cruz Biotechnology 1:100 GLI3 Rabbit Wang et al., 2000 1:100 Open in a separate window DSHB, Developmental Studies Hybridoma Bank.

Techniques: Functional Assay, Derivative Assay, Injection, Immunocytochemistry, Activation Assay, Inhibition

Journal: The Journal of Neuroscience

Article Title: Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

doi: 10.1523/JNEUROSCI.3046-14.2015

Figure Lengend Snippet: Early RA exposure yields MNs in the presence of SMO antagonist. A, Early exposure to RA both alone or in combination with Pur progressively upregulates SHH transcript levels during neural induction. B, RA induces SHH transcripts in a dose-dependent manner that correlates with RA activity as measured by RA-inducible RAR-β2 mRNA expression. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05 versus 0 μm RA, Dunnett test. C, qRT-PCR for SHH mRNA at day 21 demonstrates that very early RA exposure is required for optimal SHH upregulation. Patterning duration (2 weeks) held constant between all conditions. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05 versus RA or RA + Pur initiated at day 1, respectively, Dunnett test. D, E, Early RA exposure in the absence or presence of Pur upregulated transcripts of floor plate markers FOXA2 (D) and F-SPONDIN and NETRIN-1 (E). F, Transcripts of the floor plate markers FOXA2 and NETRIN-1 require exposure to RA or RA + Pur at very early time points during neural induction as measured by qRT-PCR. G, Immunocytochemistry at day 16 for HB9 (green) and ISL1 (red; top) and NKX6.1 (red; bottom) reveals that RA induces postmitotic and progenitor MN markers in the presence of 5 μm CUR61414. H, Quantification of markers present in G. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001 versus RA, Dunnett test. Scale bars, 10 μm. I, Immunocytochemistry at day 11 for FOXA2 demonstrates that the SMO antagonist CUR61414 (5 μm) inhibits SHH-mediated, but not RA-mediated, induction of FOXA2.

Article Snippet: Blocking was performed in PBS containing 1% BSA supplemented with 0.3% Triton for permeabilization for 30 min to 1 h. Cells were incubated in primary antibodies diluted in blocking buffer (without Triton) overnight at 4°C, followed by several washes with PBS, and then incubation in Alexa Fluor-labeled secondary antibodies (Invitrogen) diluted 1:400 in blocking buffer for 30 min to 1 h. Please refer to the antibody list in for details on sources and concentrations. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Species, isotype Company Dilution GFP Chicken Abcam 1:1000 HB9 Mouse, IgG1 DSHB 1:100 NKX6.1 Mouse, IgG1 DSHB 1:100 ISL1/2 Mouse, IgG2b DSHB 1:100 LIM3 Mouse, IgG1 DSHB 1:100 CHAT Goat Chemicon 1:100 TUJ1 Mouse or rabbit Covance 1:500 MAP2 Mouse Sigma-Aldrich 1:200 PAX6 Rabbit Covance 1:75 SMI32 Mouse Millipore 1:1000 OLIG2 Rabbit Millipore 1:200 FOXA2 Goat Santa Cruz Biotechnology 1:100 GLI3 Rabbit Wang et al., 2000 1:100 Open in a separate window DSHB, Developmental Studies Hybridoma Bank.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

doi: 10.1523/JNEUROSCI.3046-14.2015

Figure Lengend Snippet: Genetic suppression of GLI3 extends the window of competency toward MN fate. A, qRT-PCR confirms suppression of GLI3 mRNA during neural induction in DOX-treated RFP+ cells but not RFP− cells or untreated cells using our DOX-inducible shRNAmir GLI3-RFP hESC line. Data are shown as mean ± SEM; n = 3 independent experiments; **p < 0.01; *p < 0.05 versus LSB, Dunnett test. B, Diagram depicting patterning paradigm for experiment. C, Quantification of immunocytochemistry at day 21 for HB9 and ISL1 shows that GLI3 suppression via DOX treatment significantly rescues ability of RA to derive MNs from hPSCs at a later neural stage. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001; ***p < 0.001; **p < 0.01 versus RA, Dunnett test; late patterning compared with late patterning + DOX using unpaired t tests, two-tailed. D, Immunocytochemistry at day 23 for ISL1 (red), HB9 (green), and TUJ1 (blue) demonstrates the ability to specify MNs with similar efficiency to early patterning when later patterning stages are combined with DOX-induced GLI3 suppression. Scale bars, 100 μm. E, PCR amplification of GLI3 from genomic DNA of control (H9 and GLI3.3) and knock-out lines (GLI3.1 and GLI3.2) confirms a migrational shift of the PCR products from the knock-out lines consistent with genomic deletion. F, Western blot for GLI3 verifies loss of GLI3 protein in CRISPR GLI3 knock-out hESC lines (GLI3.1 and GLI3.2) but not the untargeted control line (GLI3.3). G, Immunocytochemistry at day 29 for HB9 (red), ISL1 (green), and TUJ1 (blue) shows knock-out but not control lines have the ability to specify MNs at later patterning stages with similar efficiency to early patterning of control line. H, Quantification of immunocytochemistry at day 21 for ISL1, HB9, and NKX6.1 shows that GLI3 knock-out rescues the ability of RA to derive MNs from hPSCs at a later neural stage. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001; **p < 0.01 versus late RA in GLI3.3 line, Dunnett test.

Article Snippet: Blocking was performed in PBS containing 1% BSA supplemented with 0.3% Triton for permeabilization for 30 min to 1 h. Cells were incubated in primary antibodies diluted in blocking buffer (without Triton) overnight at 4°C, followed by several washes with PBS, and then incubation in Alexa Fluor-labeled secondary antibodies (Invitrogen) diluted 1:400 in blocking buffer for 30 min to 1 h. Please refer to the antibody list in for details on sources and concentrations. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Species, isotype Company Dilution GFP Chicken Abcam 1:1000 HB9 Mouse, IgG1 DSHB 1:100 NKX6.1 Mouse, IgG1 DSHB 1:100 ISL1/2 Mouse, IgG2b DSHB 1:100 LIM3 Mouse, IgG1 DSHB 1:100 CHAT Goat Chemicon 1:100 TUJ1 Mouse or rabbit Covance 1:500 MAP2 Mouse Sigma-Aldrich 1:200 PAX6 Rabbit Covance 1:75 SMI32 Mouse Millipore 1:1000 OLIG2 Rabbit Millipore 1:200 FOXA2 Goat Santa Cruz Biotechnology 1:100 GLI3 Rabbit Wang et al., 2000 1:100 Open in a separate window DSHB, Developmental Studies Hybridoma Bank.

Techniques: Quantitative RT-PCR, Immunocytochemistry, Two Tailed Test, Amplification, Knock-Out, Western Blot, CRISPR

Journal: bioRxiv

Article Title: Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

doi: 10.1101/2022.07.05.498885

Figure Lengend Snippet: A) UMAP plots of combined human and mouse single-cell expression profiles following canonical correlation analysis (CCA)-based integration. Cells are colored based on cluster identity or expression level of key marker genes. B) The same UMAP plots as in , with only human (top) or mouse (bottom) cells colored. C) Normalized overlap between individual human or mouse clusters (rows) to the set of common clusters (columns), as defined by clustering following integration. D) Chi-square distance between human and mouse clusters. Smaller distances indicate greater similarity in overlap distribution across common clusters E) Expression dynamics of key marker genes in human and mouse shows that human NKX2-2 expression is uniquely characterized by upregulation prior to peak neurogenesis (ISL1 upregulation). F) Immunofluorescence labeling of human and mouse embryoid bodies for NKX2-2, ISL1/2 and OLIG2 across different timepoints. Human NKX2-2 is first detected at the onset of neurogenesis (day 11) (Scale bars = 50um).

Article Snippet: ISL1/2 (Mouse, 1:100 DSHB 4D5 supernatant), ISL1/2 (Goat, 1:5000 Neuromics GT15051), MNX1 (Guinea pig, 1:100 from Jessell Lab), FOXP1 (Mouse, 1:400 Santa Cruz A-2 sc-398811), NKX2-2 (Mouse, 1:100 DSHB 74.5A5 supernatant), BrdU (Rat, 1:400 Abcam ab6326), LHX1/2 (Mouse, 1:100 DSHB 4F2 supernatant), NeuN (Rabbit, 1:1000 Millipore Sigma ABN78), OLIG2 (Guinea pig, 1:100 from Jessell Lab), Ki67 (Mouse, 1:100 BDPharmingen 550609), Cleaved Caspase3 (Rabbit, 1:200 Cell Signaling #9661), LHX3 (1:100 DSHB 67.4E12 supernatant)

Techniques: Expressing, Marker, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

doi: 10.1101/2022.07.05.498885

Figure Lengend Snippet: A) The relative rates of detection (y-axis) in human-specific progenitors (vpMN) versus pMN cells shows that NKX2-2 expression is highly selective to human-specific progenitors. B) Schematic of NKX2-2-P2A-CreERT2 human iPS cell line design. C) Day 11 human cells immediately after 48 hours of 4-OHT treatment immunostained for NKX2-2 shows that the majority of RFP-positive cells are NKX2-2-positive. (Scale bar = 50um) D,E) Day 18 human cells after 4OHT treatment immunostained for pan-neuronal marker NEUN, NKX2-2, OLIG2 and MNX1. NEUN + /RFP + and MNX1 + /RFP + cells indicate that human-specific progenitors give rise to motor neurons by day 18, although many RFP + cells still remain progenitor-like (OLIG2 + and/or NKX2-2 + ). (Scale bars = 50um) F) Flow-cytometry analysis of day 11 and day 18 RFP + and RFP - cells immunostained for NKX2-2, OLIG2 and ISL1/2.

Article Snippet: ISL1/2 (Mouse, 1:100 DSHB 4D5 supernatant), ISL1/2 (Goat, 1:5000 Neuromics GT15051), MNX1 (Guinea pig, 1:100 from Jessell Lab), FOXP1 (Mouse, 1:400 Santa Cruz A-2 sc-398811), NKX2-2 (Mouse, 1:100 DSHB 74.5A5 supernatant), BrdU (Rat, 1:400 Abcam ab6326), LHX1/2 (Mouse, 1:100 DSHB 4F2 supernatant), NeuN (Rabbit, 1:1000 Millipore Sigma ABN78), OLIG2 (Guinea pig, 1:100 from Jessell Lab), Ki67 (Mouse, 1:100 BDPharmingen 550609), Cleaved Caspase3 (Rabbit, 1:200 Cell Signaling #9661), LHX3 (1:100 DSHB 67.4E12 supernatant)

Techniques: Expressing, Marker, Flow Cytometry

Journal: bioRxiv

Article Title: Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

doi: 10.1101/2022.07.05.498885

Figure Lengend Snippet: A) Varying lengths and concentrations of 4-OHT pulse affect the efficiency of recombination-based RFP expression. B) Proportions of NKX2-2-positive cells within RFP-positive populations 48 hours post 4-OHT removal. C) Flow cytometry analysis of dissociated day 16 human cultures immunostained for MNX1 and ISL1/2 shows that the vast majority of ISL1/2-positive cells are MNX1-positive and vice versa in both RFP + and RFP - populations. D) Day 18 human cultures immunostained for NEUN and ISL1/2 shows that the vast majority of NEUN-positive cells are ISL1/2 positive. (Scale bars = 50um)

Article Snippet: ISL1/2 (Mouse, 1:100 DSHB 4D5 supernatant), ISL1/2 (Goat, 1:5000 Neuromics GT15051), MNX1 (Guinea pig, 1:100 from Jessell Lab), FOXP1 (Mouse, 1:400 Santa Cruz A-2 sc-398811), NKX2-2 (Mouse, 1:100 DSHB 74.5A5 supernatant), BrdU (Rat, 1:400 Abcam ab6326), LHX1/2 (Mouse, 1:100 DSHB 4F2 supernatant), NeuN (Rabbit, 1:1000 Millipore Sigma ABN78), OLIG2 (Guinea pig, 1:100 from Jessell Lab), Ki67 (Mouse, 1:100 BDPharmingen 550609), Cleaved Caspase3 (Rabbit, 1:200 Cell Signaling #9661), LHX3 (1:100 DSHB 67.4E12 supernatant)

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

doi: 10.1101/2022.07.05.498885

Figure Lengend Snippet: A) Top enriched pathways in Reactome analysis of genes upregulated in vpMN (p-value <= 0.0001). B) Differential gene expression analysis between vpMN (H3) and pMN (H2 and H8) cells. C) Changes in the proportion of pMN (OLIG2 + , ISL1/2 - , NKX2-2 - ), MN (ISL1 + ), OPC or vpMN (NKX2-2 + , OLIG2 + , ISL1/2 - ), and NKX2-2 + cells over time in RFP-negative (left) and -positive (right) populations. RFP + cells show relative depletion of MNs, especially during early neurogenesis (day 11-12). D) Left: Log-scale histogram of RFP expression over time shows that the proportion of RFP + cells increases over time. Right: The proportion of RFP + cells (y-axis) in both total and MN populations increases in a monotonic manner. E) RFP + proportions stabilize upon DAPT addition to drive cells out of cell cycle (n.s. = non-significant; all pair-wise measurements are significantly different p <= 0.05 unless otherwise stated). F) Schematic of cumulative BrdU labeling assay to derive MN birthcurve. G) Left: Changes in the proportion of BrdU + cells within MNs over time for RFP + , total and RFP - (imputed) populations show a right-shift in the RFP + BrdU curve, indicating that RFP + remain mitotic until later stages on average. Right: Birthcurve of motor neurons in RFP + , total and RFP - (imputed) populations shows that peak neurogenesis of RFP - and RFP + cells is between days 11-12 and days 13-17, respectively. H) Left: Estimation of cell cycle length based on EdU pulse-labeling shows that RFP + /OLIG2 + /ISL1/2 - have similar cell cycle length compared to all OLIG2 + /ISL1/2 - cells. Right: RFP + and RFP - cells display similar proportions of cleaved Caspase-3-positive progenitors and motor neurons. I) Average number of motor neurons produced per mouse or human (vpMN, pMN) progenitor cell.

Article Snippet: ISL1/2 (Mouse, 1:100 DSHB 4D5 supernatant), ISL1/2 (Goat, 1:5000 Neuromics GT15051), MNX1 (Guinea pig, 1:100 from Jessell Lab), FOXP1 (Mouse, 1:400 Santa Cruz A-2 sc-398811), NKX2-2 (Mouse, 1:100 DSHB 74.5A5 supernatant), BrdU (Rat, 1:400 Abcam ab6326), LHX1/2 (Mouse, 1:100 DSHB 4F2 supernatant), NeuN (Rabbit, 1:1000 Millipore Sigma ABN78), OLIG2 (Guinea pig, 1:100 from Jessell Lab), Ki67 (Mouse, 1:100 BDPharmingen 550609), Cleaved Caspase3 (Rabbit, 1:200 Cell Signaling #9661), LHX3 (1:100 DSHB 67.4E12 supernatant)

Techniques: Expressing, Labeling, Produced

Journal: bioRxiv

Article Title: Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

doi: 10.1101/2022.07.05.498885

Figure Lengend Snippet: A ) Flow cytometry plots for days 11-16 human cultures immunostained for ISL1/2, OLIG2 and NKX2-2 shows progressive increase in ISL1/2 expression in both RFP + and RFP - populations. B) Proportions of ISL1/2 + , OLIG2 + , OLIG2 + /NKX2-2 + and NKX2-2 + cell types in day 16 cultures following DAPT treatment on day 11 shows that ~90% of both RFP + and RFP - populations are ISL1/2-positive. C) Proportion of RFP+ cells within newborn motor neuron population (as determined by BrdU labeling) increases over time. D) Day 18 human cultures with BrdU added on day 11 (left) or day 16 (right), immunolabeled for BrdU and ISL1/2. (Scale bar s= 50um) E) Relative numbers of motor neuron progenitors and motor neurons at the onset of and tail-end of neurogenesis in both mouse and human (normalized to number of progenitor cells at the onset of neurogenesis).

Article Snippet: ISL1/2 (Mouse, 1:100 DSHB 4D5 supernatant), ISL1/2 (Goat, 1:5000 Neuromics GT15051), MNX1 (Guinea pig, 1:100 from Jessell Lab), FOXP1 (Mouse, 1:400 Santa Cruz A-2 sc-398811), NKX2-2 (Mouse, 1:100 DSHB 74.5A5 supernatant), BrdU (Rat, 1:400 Abcam ab6326), LHX1/2 (Mouse, 1:100 DSHB 4F2 supernatant), NeuN (Rabbit, 1:1000 Millipore Sigma ABN78), OLIG2 (Guinea pig, 1:100 from Jessell Lab), Ki67 (Mouse, 1:100 BDPharmingen 550609), Cleaved Caspase3 (Rabbit, 1:200 Cell Signaling #9661), LHX3 (1:100 DSHB 67.4E12 supernatant)

Techniques: Flow Cytometry, Expressing, Labeling, Immunolabeling

Journal: bioRxiv

Article Title: Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

doi: 10.1101/2022.07.05.498885

Figure Lengend Snippet: A) Day 18 human cultures immunostained for LHX3, FOXP1 and ISL1/2 show that RFP+ cells are found in both MMC (LHX3 + ) as well as LMC (FOXP1 + ) cells. (Scale bars = 50um) B) The relative proportions of MMC, LMCm and LMCl subtypes in day 20 RFP + , total and RFP - (imputed) populations following late DAPT treatment on day 15 shows that RFP+ cells are enriched for LMC subtypes. C) Flow cytometry analysis of cells immunostained for ISL1/2 and FOXP1 shows temporal specification of MMC, LMCm and LMCl subtypes. D) The proportion of FOXP1 + cells among those that are born in a 24-hour, 48hour, or 16-day window, based on EdU and BrdU dual-labeling. E) Day 14 human cells treated with DAPT on day 10 show similar FOXP1 expression in RFP + and RFP - populations.

Article Snippet: ISL1/2 (Mouse, 1:100 DSHB 4D5 supernatant), ISL1/2 (Goat, 1:5000 Neuromics GT15051), MNX1 (Guinea pig, 1:100 from Jessell Lab), FOXP1 (Mouse, 1:400 Santa Cruz A-2 sc-398811), NKX2-2 (Mouse, 1:100 DSHB 74.5A5 supernatant), BrdU (Rat, 1:400 Abcam ab6326), LHX1/2 (Mouse, 1:100 DSHB 4F2 supernatant), NeuN (Rabbit, 1:1000 Millipore Sigma ABN78), OLIG2 (Guinea pig, 1:100 from Jessell Lab), Ki67 (Mouse, 1:100 BDPharmingen 550609), Cleaved Caspase3 (Rabbit, 1:200 Cell Signaling #9661), LHX3 (1:100 DSHB 67.4E12 supernatant)

Techniques: Flow Cytometry, Labeling, Expressing

Journal: bioRxiv

Article Title: Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

doi: 10.1101/2022.07.05.498885

Figure Lengend Snippet: A) Left: Day 14 human cultures immunostained for FOXP1 and LHX1 shows that LHX1 is not yet expressed, but by day 18 is robustly expressed and overlaps with FOXP1, indicating that these cells are likely LMCl subtypes (middle). Right: LHX1-positive cells have lower ISL1/2 expression, further indicating that they are LMCl motor neurons. (Scale bars = 50um)

Article Snippet: ISL1/2 (Mouse, 1:100 DSHB 4D5 supernatant), ISL1/2 (Goat, 1:5000 Neuromics GT15051), MNX1 (Guinea pig, 1:100 from Jessell Lab), FOXP1 (Mouse, 1:400 Santa Cruz A-2 sc-398811), NKX2-2 (Mouse, 1:100 DSHB 74.5A5 supernatant), BrdU (Rat, 1:400 Abcam ab6326), LHX1/2 (Mouse, 1:100 DSHB 4F2 supernatant), NeuN (Rabbit, 1:1000 Millipore Sigma ABN78), OLIG2 (Guinea pig, 1:100 from Jessell Lab), Ki67 (Mouse, 1:100 BDPharmingen 550609), Cleaved Caspase3 (Rabbit, 1:200 Cell Signaling #9661), LHX3 (1:100 DSHB 67.4E12 supernatant)

Techniques: Expressing