rabbit anti‐en1  (Absolute Biotech Inc)

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Triple RNAscope in situ hybridization showing Engrailed‐1 ( En1 ), Choline acetyltransferase ( ChAT ), and Calbindin‐1 ( Calb1 ) expression in the lumbar spinal cord. En1 is expressed in V1 interneurons, dorsal (V1 P ) and ventral (V1 R ) to the main ChAT ‐expressing motoneuron pool. Ventral interneurons correspond to Renshaw cells as shown by Calb1 expression. Scale bar: 500 μm. RT–qPCR of RNA from the lumbar enlargement at 4.5 and 16 months of age shows stable En1 expression in WT at both ages and a twofold reduction of expression in heterozygous mice. Unpaired two‐sided t ‐test. ** P < 0.005; *** P < 0.0005. n = 3. Values are mean ± SD. Triple staining EN1 IHC (green), En1 RNAscope ISH (red), and ChAT RNAscope (blue) demonstrating the double staining of En1 mRNA and protein (EN1) in the V1 interneuron population (left panel insets, arrowheads point toward examples of double‐stained V1 interneurons), and the presence of EN1 protein in large cells not expressing En1 mRNA (left panel) but expressing ChAT (right panel insets). Scale bar: 500, 30 μm for high magnification insets. Left: EN1 (red) detected with the LSBio antibody is localized in ChAT‐expressing neurons (green) in the ventral horns of the spinal cord. Right: EN1 signal is lost upon preincubation of the antibody with 1.5 M excess of recombinant hEN1. Scale bar: 50 μm. Left: Relative positions of the different oligonucleotides selected to genotype the E15 embryos and examples of the genotyping based on the combination of PCRs with the different pairs of primers. Right: Double ChAT/EN1 immunostaining demonstrating the co‐localization of the two proteins in the WT and the En1 ‐Het ventral cord and the absence of EN1 staining in MNs from En1 ‐KO embryos. Note that the staining is reduced in En1 ‐Het embryos, compared with WT embryos. Data information: These experiments were performed once. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: In Situ Hybridization, Expressing, Quantitative RT-PCR, Staining, Double Staining, Recombinant, Immunostaining

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Western blots of spinal cord (SC) and ventral midbrain (VMB) extracts demonstrating that the 86/8 and LSBio antibodies recognize in both structures the same protein migrating with recombinant EN1 velocity. No staining is observed in the absence of primary antibody (left panel). This experiment was performed twice. Double staining of 3‐month‐old WT and En1 ‐Het ventral MNs with the anti‐ChAT antibody and the anti‐EN1 LSBio antibody at various dilutions. EN1 staining decreases with increasing dilutions of the antibody. The loss of staining is more rapid in En1 ‐Het than in WT mice. Scale bar = 50 μm. Data information: This experiment was performed once.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Western Blot, Recombinant, Staining, Double Staining

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Analysis of the number of En1 +, Calb1 +, and ChAT + neurons (triple RNAscope). At 4.5 months, WT and En1 ‐Het mice show no difference in the number of cells expressing En1 , Calb1 , or ChAT . Unpaired two‐sided t ‐test. n = 5–6. Cresyl violet and ChAT staining of a ventral WT spinal cord at the lumbar level. Scale bar: 100 μm. Cresyl violet and ChAT staining at the lumbar level show no medium‐size (200–299 μm 2 ) cell loss (γMNs) and a decrease of about 50% in the number of large‐size (> 300 μm 2 ) cells (αMNs). Unpaired two‐sided t ‐test. ** P < 0.005; **** P < 0.0001. n = 5. Lumbar level Cresyl violet staining shows that, in contrast with small‐ and medium‐size neurons (interneurons and γMNs, left and center panels), large‐size neurons (αMNs, right panel) undergo progressive death first measured at 4.5 months. The values represent the average number of cells per ventral horn. For the small neurons (100–199 μm 2 ), there was no main effect, two‐way ANOVA for repeated measures for treatment group: F (1, 43) = 0.0017, P = 0.968, ns. For the medium‐sized neurons (200–299 μm 2 ) two‐way ANOVA for repeated measures for treatment group: showed no main effect F (1, 43) = 2.085, ns. For the large neurons (> 300 μm 2 ), two‐way ANOVA for repeated measures showed a significant main effect F (1, 43) = 59.99, P < 0.0001. Post hoc comparisons were performed by unpaired two‐sided t ‐test with equal SD comparing WT with En1‐ Het at each time point (* P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001). n = 5–6. Compared with WT mice, En1 ‐Het mice experience gradual strength loss. This loss is observed with the forepaw grip strength (left panel), the inverted grid test (center panel) and the hindlimb extensor reflex test (right panel). Strength loss is first observed between 2 and 3 months, thus before measurable αMN cell body loss. Two‐way ANOVA showed significant main effects for grip strength ( F (1, 136) = 19.18, P < 0.0001), inverted grid test ( F (1, 103) = 143.1, P < 0.0001), and extensor score ( F (1, 103) = 10.1, P < 0.0001). Comparisons were made by unpaired two‐sided t ‐test with equal SD comparing WT with En1‐ Het at each time point (* P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001). n = 4–20. Data information: The analysis of neuron number in (A), the direct comparison of Cresyl violet and ChAT in (C), and the longitudinal studies in (D and E) were performed once. Values are mean ± SD. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Expressing, Staining

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Analysis of EN1 amount in MNs at 3 months in WT and En1 ‐Het mice. EN1 content is reduced by about half in γMNs (left panel) and αMNs (right panel). EN1 was revealed by the LSBio antibody allowing for the visualization of endogenous EN1. Unpaired two‐sided t ‐test with equal SD (** P < 0.005; n = 5). Values are mean ± SD. Quantification of EN1 amount in γMNs and αMNs with the LSBio antibody at 3, 4.5 and 9 months in WT and En1 ‐Het mice. Values at 3 months correspond to the ones shown in panel A. At 4.5 and 9 months, the amount of EN1 in MNs is similar in En1 ‐Het and WT mice suggesting that, with time, each remaining MN receives a higher amount of EN1. Two‐way ANOVA showed a significant main effect for the γMNs ( F (1, 20) = 16.93, P = 0.0005) and αMNs ( F (1, 20) = 19.03, P = 0.0003). Unpaired two‐sided t ‐test with equal SD (* P < 0.05; n = 4–5). Data information: This experiment was performed once. Values are mean ± SD. Hypothetical representation of EN1 availability to αMNs in WT and Het mice. In the En1 ‐Het mouse, each V1 interneurons only provides half as much EN1 to the full population of αMNs at 3 months of age. At 4.5 months of age and later, half of the αMNs have been lost allowing each remaining αMN to receive its full complement of EN1 from the V1 interneurons.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques:

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: NMJs of En1‐ Het and WT mice show similar numbers of AChR clusters (left panel) and late‐occurring (15.5 months) decrease in endplate area (center panel) and percentage of perforated endplates (right panel). The number of AChR clusters seems to decrease between 2 and 3 months, but two‐way ANOVA showed no significant genotype effect ( F (1, 47) = 0.1291, ns). There was a significant main effect for endplate area ( F (1, 47) = 5.778, P = 0.0202) and for the percentage of perforated endplates ( F (1, 47) = 13.82, P = 0.0005). Post hoc analysis revealed significant genotype differences at 15.5 month of age. Unpaired two‐sided t ‐test with equal SD comparing WT with En1‐ Het at each time point (** P < 0.005). n = 4–8. Left panel illustrates the use of Alexa Fluor 488‐conjugated α‐bungarotoxin (α‐BTX, in green) and of neurofilament and synaptic vesicle glycoprotein antibodies (2H3 and SV2A, in red) to evaluate the percentage of fully occupied endplates (> 80% occupancy). The right panel shows that the % of fully occupied endplates decreases progressively in the En1 ‐Het mouse, starting between 3 and 4.5 months of age. Scale bar: 50 μm. Two‐way ANOVA showed a significant main effect ( F (1, 47) = 45.45, P < 0.0001). Unpaired two‐sided t ‐test with equal SD comparing WT with En1‐ Het at each time point (* P < 0.05; ** P < 0.005). n = 4–8. See details of analysis in the section. Data information: The longitudinal study in (A and B) was performed once. Values are mean ± SD. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques:

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: RT–qPCR of RNA from the lumbar enlargement at 4.5 months of WT and En1 ‐Het mice and from WT muscle. En1 expression is absent from the muscle of WT mice. Unpaired two‐sided t ‐test with equal SD (*** P < 0.005; **** P < 0.0005; n = 4–5). Values are mean ± SD. Immunohistochemistry for EN1 protein (LSBio antibody, in green) shows its absence at the level of the NMJ (α‐BTX, in red). Scale bar = 50 μm. Data information: This experiment was done once. Values are mean ± SD.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Experimental paradigm and structure of AAV8‐encoded constructs containing glial fibrillary acidic protein (GFAP) promoter for expression in astrocytes, Immunoglobulin K (IgK) signal peptide for secretion, anti‐ENGRAILED single‐chain antibody (scFvEN1), 6 myc tags (6xMyc), skipping P2A peptide, and enhanced Green Fluorescent Protein (eGFP). An inactive control antibody (scFvMUT) contains a cysteine to serine mutation that prevents disulfide bond formation between IgG chains, thus epitope recognition. The AAV8 was injected in 1‐month‐old WT mice, and the strength phenotypes were followed for 6 months before anatomical analysis. Analysis of 1‐month postinjection showing that scFv antibodies are expressed in astrocytes (white arrowhead) double‐stained for GFAP and Myc and exported (empty arrowhead). Scale bar: 50 μm. Left panel illustrates that expressing the scFvEN1, but not scFvMUT, abolishes EN1 staining by LSBio anti‐EN1 antibody in ventral horn ChAT+ cells and right panel quantifies this inhibition 1‐way ANOVA followed by Tukey corrected post hoc comparisons ( n = 5 mice per group, *** P < 0.0005, **** P < 0.0001). Scale bar: 100 μm. The three graphs illustrate how the WT antibody but not its mutated version leads to progressive strength decrease. Two‐way ANOVA showed significant main effects for grip strength ( F (2, 12) = 15.88, P < 0.0005), inverted grid ( F (2, 107) = 19.86, P < 0.0001), and extensor score ( F (2, 12) = 30.22, P < 0.0001) followed by Tukey corrected post hoc comparisons (* P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001). n = 5 per treatment. Six months following infection (7‐month‐old mice), extracellular EN1 neutralization does not modify the number of AChR clusters, nor the endplate surface area, nor the percentage of perforated endplates. In contrast, the percentage of fully occupied endplates is diminished (right end panel). 1‐way ANOVA followed by Tukey corrected post hoc comparisons (** P < 0.005). n = 5 per treatment. Six months following infection, extracellular EN1 neutralization does not globally modify the total neuron number of cells at the lumbar level (left panel). A separate analysis of small (100–199 μm 2 ), medium (200–299 μm 2 ), and large (>300 μm 2 ) neurons demonstrate a specific ( P < 0.0557) loss of the latter category (αMNs). 1‐way ANOVA followed by Tukey corrected post hoc comparisons. N = 5 per treatment. Data information: The extracellular neutralization study was performed twice. Values are mean ± SD. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Construct, Expressing, Mutagenesis, Injection, Staining, Inhibition, Infection, Neutralization

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Comparison between En1‐het mouse and scFvEN1 models. Data and graphs are from main figures (primarily Figs and ). WT mice injected with scFvEN1 show similar results to those obtained in the En 1‐Het mouse with a milder strength loss, a smaller decrease in the number of fully occupied endplates, and the specific loss of large‐size αMNs. At 7 months, scFvEN1 injected mice have a phenotype similar to 3‐month‐old En1 ‐Het mice.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Injection

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Mice were tested for strength at 3 months of age, before the onset of αMN loss, but when strength has already decreased in En1 ‐het mice as measured in the forepaw grip strength, inverted grid, and extensor reflex tests (left side of each graph). The next day, the En1 ‐het mice were separated into two groups. One group received buffer and the other group recombinant hEN1 (1 μg in 5 μl), injected at the L5 level. One and a half months later (4.5 months of age) En1 ‐het mice injected with hEN1 have recovered normal strength, in contrast with noninjected mice or mice injected with buffer. Unpaired two‐sided t ‐test used at 3 months of age. **** P < 0.0001. For 4.5‐month comparisons, 1‐way ANOVA followed by Tukey corrected post hoc comparisons. *** P < 0.0005; **** P < 0.0001. n = 9–31. At 4.5 months, following hEN1 injection at 3 months, the percentage of fully occupied endplates and the number of αMNs are not significantly different from control values. 1‐way ANOVA followed by Tukey corrected post hoc comparisons. *** P < 0.0005; **** P < 0.0001. n = 5–21. Data information: The weakness and reversal with hEN1 and the neuroprotection were replicated in five independent experiments. Values are mean ± SD. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Recombinant, Injection

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Top left panel shows the single injection protocol whereby hEN1 is injected at 3 months (1 μg in 5 μl) intrathecally at the L5 level and mouse behavior followed for 24 weeks. The 3 time‐course graphs demonstrate that a single injection restores strength measured by the tests of grip strength, time on the inverted grid and extensor reflex and that this effect lasts for 12 weeks. After 12 weeks, strength decreases progressively but, even after 24 weeks, remains superior to that of untreated En1 ‐Het mice. At 24 weeks, the % of fully occupied endplates is inferior to that of WT mice, but superior to that of noninjected En1 ‐Het mice. The same holds true for the number of αMNs. Two‐way ANOVA revealed significant main effects for grip strength ( F (1, 76) = 143.6, P < 0.0001), inverted grid ( F (1, 76) = 128.6, P < 0.0001), and extensor score ( F (1, 76) = 34.91, P < 0.0001). At the different times, the groups were compared by unpaired t‐test with equal variances comparing WT with En1‐ Het injected at each time point. (* P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001). For the endplate analysis and αMNs, groups were compared by 1‐way ANOVA followed by Tukey corrected post hoc comparisons (* P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001). n = 4–10. Based on the results shown in A, a new experiment was performed with a second injection 12 weeks after the first injection at 3 months of age. Again, the injection at 3 months of age restored strength and in mice receiving the second injection strength was maintained an additional 10 weeks or more compared with mice receiving a single injection. The three strength graphs demonstrate a positive effect of the second injection with values intermediate between those measured in WT mice and En1‐Het mice with a single injection. The percentage of fully occupied endplates and the number of αMNs are back to wild‐type values in En1 ‐Het mice injected twice. Two‐way ANOVA revealed significant main effects for grip strength ( F (2, 81) = 25.47, P < 0.0001), inverted grid ( F (2, 91) = 51.96, P < 0.0001), and extensor score ( F (2, 104) = 30.42, P < 0.0001). At all times, groups were compared by unpaired t‐test with equal variances through 12 weeks. After, the groups were compared by Tukey corrected post hoc comparisons (* P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001). For the endplate analysis and αMNs, groups were compared by 1‐way ANOVA followed by Tukey corrected post hoc comparisons (* P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001). n = 4–10. Data information: The single injection and two injection time‐course studies were performed once each. Values are mean ± SD. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Injection

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Top left panel shows that 24 h after intrathecal injection (1 μg in 5 μl) at the L5 level of 2‐month‐old mice, hEN1 (red) can be primarily visualized in ventral horn ChAT+ cells (green). Arrowheads show hEN1 internalized by MNs. Scale bar: 100 μm. Top right panels show the progressive accumulation and clearance of hEN1 in ventral horn MNs, with a peak between 6 and 24 h. EN1 was revealed by the 86/8 antibody allowing for the visualization of exogenous EN1 only (scale bar: 100 μm). Bottom panels show the quantification of EN1 in γMNs and αMNs with the 86/8 (two left panels) and the LSBio (two right panels) antibodies. Since the LSBio sees both endogenous and exogenous EN1, the increase is only threefold, but qualitatively, the results are very similar, demonstrating rapid internalization and clearance of the exogenous protein and allowing one to calculate a half‐life of 24 h. One‐way ANOVA followed by Tukey corrected post hoc comparisons (* P < 0.05; ** P < 0.005; *** P < 0.0005). When not significant, P ‐values are shown. n = 3. Values are mean ± SD. Data information: The internalization experiments were performed once with each antibody. Left panel gives examples of putative glycosaminoglycan (GAG)‐binding domain in 11 homeoprotein transcription factors. Based on the alignment and on published work on OTX2 (Beurdeley et al , ) and EN2 (preprint: Cardon et al , ), a putative EN1 GAG‐binding domain (RK‐EN1) was designed. Middle panel quantifies the inhibitory effect of RK‐EN1 on hEN1 capture (86/8 antibody) by ChAT+ cells demonstrating that RK‐EN1 in a 1 to 20 ratio reduces the % of EN1‐positive MNs (ChAT+) from 50 to less than 10%. The right panel demonstrates that this inhibitory activity is not shared by the mutant AA peptide or by a scrambled (Scr) peptide. Unpaired two‐sided t ‐test with equal SD (** P < 0.005; *** P < 0.0005). n = 2–5. Data information: The GAG competition experiment was performed twice as described in the text. Values are mean ± SD except for conditions with two observations for which both data points are shown. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Injection, Binding Assay, Activity Assay, Mutagenesis

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: The search for genes differentially expressed in WT and En1 ‐Het mDA neurons and expressed in MNs, thus putative non‐cell‐autonomous EN1 targets in MNs, allowed for the identification of 402 genes (after pathway selection). These genes were investigated for an interaction with genes mutated in the main 4 familial ALS forms. Among them, p62/SQSTM1 ( p62 ) expression is upregulated in the SNpc (RNA‐seq) and in MNs of En1 ‐Het mice. Immunohistochemical staining shows the presence of high amounts of p62/SQSTM1 in ChAT+ cell bodies of 3‐month‐old mice. Scale bar: 50 μm. Intensity measurements demonstrate that the mean of p62/SQSTM1 expression increases with age in WT γMNs (left) and αMNs (right). However, comparing WT and En1 ‐Het shows a significant difference only at 3 months and not later. Unpaired two‐sided t ‐test. (* P < 0.05; ** P < 0.005; *** P < 0.0005, **** P < 0.0001). Data information: This experiment was done once. Values are mean ± SD. Between 151 and 1,215 neurons and 3–5 mice were analyzed for each condition. Mean intensity of p62/SQSTM1 expression is increased in γMNs (left) and αMNs (right) in mice expressing scFvEN1 6 months after virus injection (7‐month‐old mice) demonstrating that EN1 extracellular neutralization increases p62/SQSTM1 expression. Expression of the mutated antibody (scFvMUT) does not increase p62/SQSTM1 expression. Unpaired two‐sided t ‐test. (* P < 0.05; ** P < 0.005; *** P < 0.0005, **** P < 0.0001). Data information: This experiment was performed once. Values are mean ± SD. Between 448 and 759 neurons and 5 mice were analyzed for each condition. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Selection, Expressing, RNA Sequencing Assay, Immunohistochemical staining, Staining, Injection, Neutralization

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: Left panel: Injection and analysis protocol. Right panel: muscle strength analysis demonstrating that hEN1 injection at 1 month prevents muscular strength decrease observed in 3‐month‐old En1‐Het mice. One‐way ANOVA followed by two‐sided t ‐test. (* P < 0.05, **** P < 0.0001). N = 6–10. Data information: This experiment was performed once. Values are mean ± SD. Left panel: Increased p62/SQSTM1 staining in 3‐month‐old ChAT+ MNs from control En1 ‐Het is abolished by hEN1 injection at 1 month. Right panel: quantification of p62/SQSTM1 staining in γMNs and αMNs of control and hEN1‐injected 3‐month‐old En1 ‐het mice. One‐way ANOVA followed by two‐sided t ‐test. (** P < 0.01, **** P < 0.0001). Data information: This experiment was performed once. Values are mean ± SD. Between 111 and 303 neurons and 4–5 mice were analyzed for each condition. Source data are available online for this figure.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Injection, Staining

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet: EN1 transcribed and synthesized in V1 (pink) has a cell‐autonomous activity in these cells contributing to the expression of several V1 factors (FA, FB, …) that may signal to MNs (yellow) and other cell types (blue). All the latter F factors do not necessarily require EN1 activity. EN1 after its secretion by V1 interneurons is preferentially internalized by MNs (fat red arrow) and less by other cell types (thin red arrow), if at all. These other cell types express other F Factors (FX, FY, …) which are secreted and are, or not, under non‐cell‐autonomous EN1 activity. As a result, MNs experience the signaling activity of F factors (possibly EN1‐dependent as in the scheme, but not necessarily so) and internalize EN1. Following internalization, EN1 can work alone or in synergy with the F factors to regulate transcription, translation, and chromatin conformation within MNs.

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Synthesized, Activity Assay, Expressing

Journal: EMBO Reports

Article Title: ENGRAILED ‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

doi: 10.15252/embr.202256525

Figure Lengend Snippet:

Article Snippet: Rabbit anti‐EN1 LSBio , CliniSciences , LS‐B9070.

Techniques: Multiplex Assay, RNA Extraction, BIA-KA, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . Triple RNAscope in situ hybridization showing Engrailed-1 ( En1 ), Choline acetyltransferase ( ChAT ) and Calbindin-1 ( Calb1 ) expression in the lumbar spinal cord. En1 is expressed in V1 interneurons, dorsal (V1 P ) and ventral (V1 R ) to the main ChAT -expressing motoneuron pool. Ventral interneurons correspond to Renshaw cells as shown by Calb1 expression. Scale bar: 500μm. B . RT-qPCR of RNA from the lumbar enlargement at 4.5- and 16-months of age shows stable En1 expression in WT at both ages and a two-fold reduction of expression in heterozygous mice. Unpaired t-test. **p<0.005; ***p<0.0005. n=3. C . Triple staining EN1 IHC (green), En1 RNAscope ISH (red) and ChAT RNAscope (blue) demonstrating the double-staining of En1 mRNA and protein (EN1) in the V1 interneuron population (left panel insets), and the presence of EN1 protein in large cells not expressing En1 mRNA (left panel) but expressing ChAT (right panel insets). Scale bar: 500µm or 30µm for high magnification insets. D . Left panel: Western blots of spinal cord (SC) and ventral midbrain (VMB) extracts demonstrating that the 86/8 and LSBio antibodies recognize in the 2 structures the same protein migrating with recombinant EN1 velocity. Right panel: Immunoprecipitation of spinal cord extracts by LSBio or 86/8 antibody followed by Western blotting with 86/8 or LSBio antibody, respectively. E . Left: EN1 (red) detected with the LSBio antibody is localized in ChAT-expressing neurons (green) in the ventral horns of the spinal cord. Right: EN1 signal is lost upon preincubation of the antibody with 1.5 M excess of recombinant hEN1. Scale bar: 50µm.

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: In Situ Hybridization, Expressing, Quantitative RT-PCR, Staining, Double Staining, Western Blot, Recombinant, Immunoprecipitation

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . Experimental paradigm and structure of AAV8-encoded construct containing glial fibrillary acidic protein (GFAP) promoter for expression in astrocytes, Immunoglobulin K (IgK) signal peptide for secretion, anti-ENGRAILED single-chain antibody (scFvEN1), 6 myc tags (6xMyc), skipping P2A peptide and enhanced Green Fluorescent Protein (EGFP). An inactive control antibody (scFv MUT) contains a cysteine to serine mutation that prevents disulfide bond formation between IgG chains, thus epitope recognition. The AAV8 was injected in 1-month-old WT mice and the strength phenotypes were followed for 6 months before anatomical analysis. B . Analysis 1 month post-injection showing that scFv antibodies are expressed in astrocytes (white arrowhead) double-stained for GFAP and Myc and exported (empty arrowhead). Scale bar: 50 µm. C . Left panel illustrates that expressing the scFvEN1, but not scFvMUT, abolishes EN1 staining by LSBio anti-EN1 antibody in ventral horn ChAT+ cells and right panel quantifies this inhibition 1-way ANOVA followed by Tukey corrected post-hoc comparisons. (n=5 mice per group, ***p<0.0005, ****p<0.0001). Scale bar: 100µm. D . The three graphs illustrate how the WT antibody but not its mutated version leads to progressive neuromuscular strength decrease. 1-way ANOVA followed by Tukey corrected post-hoc comparisons. (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=5 per treatment. E . Six months following infection (7-month-old mice), extracellular EN1 neutralization does not modify the number of AChR clusters, nor the endplate surface area, nor the percentage of perforated endplates. In contrast, the percentage of fully occupied endplates is diminished (right end panel). 1-way ANOVA followed by Tukey corrected post-hoc comparisons. (**p<0.005). n=5 per treatment. F . Six months following infection, extracellular EN1 neutralization does not globally modify the total neuron number of cells at the lumbar level (left panel). A separate analysis of small (100-199 µm 2 ), medium (200-299 µm 2 ) and large (>300 µm 2 ) neurons demonstrate a specific (p<0.0557) loss of the latter category (αMNs). 1-way ANOVA followed by Tukey corrected post-hoc comparisons. n=5 per treatment.

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: Construct, Expressing, Mutagenesis, Injection, Staining, Inhibition, Infection, Neutralization

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . Analysis of the number of En1 +, Calb1 + and ChAT + neurons (triple RNAScope). At 4.5 months, WT and En1 -Het mice show no difference in the number of cells expressing En1, Calb1 or ChAT . n=5-6. B . Cresyl violet and ChAT staining of a ventral WT spinal cord at the lumbar level. Scale bar: 100 µm. C . Cresyl violet and ChAT staining at the lumbar level show no medium size (200-299 µm 2 ) cell loss (γMNs) and an about 50% decrease in the number of large size (>300 µm 2 ) cells (αMNs). Unpaired t-test. **p<0.005; ***p<0.0005. n=5. D . Lumbar level Cresyl violet staining shows that, in contrast with small and medium size neurons (interneurons and γMNs, left and center panels), large size neurons (αMNs, right panel) undergo progressive death first measured at 4.5 months. Unpaired t-test with equal SD comparing WT with En1- Het at each time point (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=5 to 6. E . Compared to WT mice, En1 -Het mice experience gradual neuromuscular strength loss. This loss is observed with the forepaw grip strength (left panel), the inverted grid test (center panel and the hindlimb extensor reflex test (right panel). Strength loss is first observed between 2 and 3 months, thus before measurable αMN cell body loss. Unpaired T-test with equal SD comparing WT with En1- Het at each time point (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=4 to 20.

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . NMJs of En1- Het and WT mice show similar numbers of AChR clusters (left panel) and late-occurring (15.5 months) decrease in endplate area (center panel) and percentage of perforated endplates (right panel). The number of AChR clusters seems to decrease between 2 and 3 months, but a 1-way ANOVA followed by Tukey corrected post hoc comparisons show that this is not significant and reflects a high variance in the analysis (p=0.071). B . Left panel illustrates the use of Alexa Fluor 488-conjugated α-bungarotoxin (α-BTX, in green) and of neurofilament and synaptic vesicle glycoprotein antibodies (2H3 and SV2A, in red) to evaluate the percentage of fully occupied endplates (>80% occupancy). The right panel show that the % of fully occupied endplates decreases progressively in the En1 -Het mouse, starting between 3 and 4.5 months of age. Scale bar: 100µm. Unpaired T-test with equal SD comparing WT with En1- Het at each time point (*p<0.05; **p<0.005). n=4 to 8.

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques:

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . Mice were tested for neuromuscular strength at three months of age when (before the onset of αMN loss), strength has already decreased in En1 -het mice as measured in the forepaw grip strength, inverted grid and extensor reflex tests (left side of each graph). The next day the En1 -het mice were separated into two groups. One group received buffer and the other group recombinant hEN1 (1 µg in 5 µl), injected at the L5 level. One and a half months later (4.5 months) En1 -het mice injected with hEN1 have recovered normal strength, in contrast with non-injected mice or mice injected with buffer. Unpaired t-test used at three months of age. ****p<0.0001. For 4.5-month comparisons, 1-way ANOVA followed by Tukey corrected post-hoc comparisons. ***p<0.0005; ****p<0.0001. n=9 to 31 B . At 4.5 months, following hEN1 injection at 3 months, the percentage of fully occupied endplates and the number of αMNs are not significantly different from control values. 1-way ANOVA followed by Tukey corrected post hoc comparisons. ***p<0.0005; ****p<0.0001. n=5 to 21

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: Recombinant, Injection

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . Top left panel shows the single injection protocol whereby hEN1 is injected at 3 months (1µg in 5µl) intrathecally at the L5 level and mouse behavior followed for 24 weeks. The 3 time course graphs demonstrate that a single injection restores neuromuscular strength measured by the tests of grip strength, time on the inverted grid and extensor reflex and that this effect lasts for 12 weeks. After 12 weeks strength decreases progressively but, even after 24 weeks, remains superior to that of untreated En1 -Het mice. At 24 weeks, the % of fully occupied end-plates is inferior to that of WT mice, but superior to that of non-injected En1 -Het mice. The same holds true for the number of αMNs. For behavior, the groups were compared by unpaired T-test with equal variances comparing WT with En1- Het injected at each time point. (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). For the endplate analysis and αMNs, groups were compared by 1-way ANOVA followed by Tukey corrected post-hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n= 4 to 10. B . Based on the results shown in A, a new experiment was performed with a second injection 12 weeks after the first injection at 3 months of age. Again, the injection at three months of age restored neuromuscular strength and in mice receiving the second injection strength was maintained an additional ten weeks or more compared to mice receiving a single injection. The three strength graphs demonstrate a positive effect of the second injection with values intermediate between those measured in WT mice and En1-Het mice with a single injection. The percentage of fully occupied endplates and the number of αMNs are back to wild type values in En1 -Het mice injected twice. Groups were compared by Unpaired T-test with equal variances comparing WT with En1- Het injected once or twice at each time point. For behavior, the groups were compared by unpaired T-test with equal variances through 12 weeks. After, the groups were compared by 1-way ANOVA and if significant followed by Tukey corrected post-hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). For the endplate analysis and αMNs, groups were compared by 1-way ANOVA followed by Tukey corrected post hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n= 4 to 10.

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: Injection

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . Top left panel shows that 24 hours after intrathecal injection (1 μg in 5µL) at the L5 level of 2-month-old mice, hEN1 (red) can be primarily visualized in ventral horn ChAT+ (green) cells Arrowheads show hEN1 internalized by MNs. Scale bar: 100 µm. Top right panels show the progressive accumulation and clearance of hEN1 in ventral horn MNs, with a pick between 6 and 24 hours. EN1 was revealed by the 86/8 antibody allowing for the visualization of exogenous EN1 only (scale bar: 100µm). Bottom panels show the quantification of EN1 in in γMNs and αMNs with the 86/8 antibody at 6, 24, 48 and 72 hours (two left panels). In the two right panels quantification is done with the LSBio antibody in non-injected mice and at 24 and 72 hours after injection. Since the LSBio sees both endogenous and exogenous EN1, the increase is only 2-fold, but qualitatively, the results are very similar, demonstrating rapid internalization and clearance of the exogenous protein and allowing one to calculate a half-life of 24 hours. 1-way ANOVA followed by Tukey corrected post-hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005). When not significant, p values shown. B . Left panel gives examples of putative glycosaminoglycan (GAG)-binding domain in 11 homeoprotein transcription factors. Based on the alignment and on published work on OTX2 and EN2 , a putative EN1 GAG-binding domain (RK-EN1) was designed. Middle panel quantifies the inhibitory effect of RK-EN1 on hEN1 capture (86/8 antibody) by ChAT+ cells demonstrating that RK-EN1 in a 1 to 20 ratio reduces the % of EN1-postive MNs (ChAT+) from 50 to less that 10%. The right panel demonstrates that this inhibitory activity is not shared by the mutant AA peptide or by a scrambled (Scr) peptide. Unpaired T-test with equal SD (**p<0.005; ***p<0.0005). n=3-5

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: Injection, Binding Assay, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: A . The search for genes differentially expressed in WT and En1 -Het mDA neurons and expressed in MNs, thus putative non-cell autonomous EN1 targets in MNs, allowed for the identification of 402 genes (after pathway selection). These genes were investigated for an interaction with genes mutated in the main 4 familial ALS forms. Among them, SQTSM1/p62 ( p62 ) expression is upregulated in the SNpc (RNA-seq) and in MNs of En1 -Het mice. B . Immunohistochemical staining shows the presence of high amounts of p62 in ChAT+ 3-month-old cell bodies. Scale bar: 50 µm. C . Intensity measurements demonstrates that the mean of SQTSM1/p62 expression increases with age in WT γMNs (left) and αMNs (right). However, comparing WT and En1 -Het shows a significant difference only at 3 months and not later. Dispersion also increases with time (except between 3- and 4.5-month-old γMNs) and differences between WT and En1 -Het are seen at the 3 ages in all MNs, except at 9 months for αMNs. Statistical analysis is explained under material and methods. D . Mean intensity and dispersion of SQTSM1/p62 expression are increased in γMNs (left) and αMNs (right) in mice expressing scFvEN1 6 months after virus injection (7-month-old mice) demonstrating that EN1 extracellular neutralization increases SQTSM1/p62 expression and dispersion. The mutated antibody (scFVMUT) does not increase p62 expression, but has a small but significant effect on dispersion.

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: Selection, Expressing, RNA Sequencing Assay, Immunohistochemical staining, Staining, Injection, Neutralization

Journal: bioRxiv

Article Title: ENGRAILED-1 transcription factor exerts a paracrine neurotrophic activity on adult spinal cord α-motoneurons

doi: 10.1101/2022.08.16.504081

Figure Lengend Snippet: EN1 transcribed and synthesized in V1 (pink) has a cell autonomous activity in these cells contributing to the expression of several V1 factors (F A , F B , …) that may signal to MNs (yellow) and other cell types (blue). All the latter F factors do not necessarily require EN1 activity. EN1 after its secretion by V1 interneurons is preferentially internalized by MNs (fat red arrow) and less by other cell types (thin red arrow), if at all. These other cell types express other F Factors (F X , F Y , …) which are secreted and are, or not, under non-cell autonomous EN1 activity. As a result, MNs experience the signaling activity of F factors (possibly EN1-dependent as in the scheme, but not necessarily so) and internalize EN1. Following internalization, EN1 can work alone or in synergy with the F factors to regulate transcription, translation and chromatin confirmation within MNs.

Article Snippet: After 30 minutes at RT in 100μM glycine buffer, 10% Normal Goat Serum (NGS, Invitrogen) or Fetal Bovine Serum (FBS, Gibco) was added in the presence of 1% Triton before incubation with primary antibodies (sheep anti-Choline Acetyltransferase (ChAT) ABCAM 1:1000, goat anti-ChAT Millipore 1:500, rabbit anti-EN1 86/8 1:300 , rabbit anti-EN1 LSBio (CliniSciences) 1:200, mouse anti-neurofilament 165kDa Developmental Studies Hybridoma Bank 1:50, mouse anti-synaptic vesicle glycoprotein 2A DSHB 1:100 and rabbit anti-p62 ABCAM 1:1000 overnight at 4°C, washed and further incubated with secondary antibodies for 2 hours at RT.

Techniques: Synthesized, Activity Assay, Expressing

Journal: STAR Protocols

Article Title: Generation of human induced pluripotent stem cell-derived cerebral organoids for cellular and molecular characterization

doi: 10.1016/j.xpro.2022.101173

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti-EN1 (1:25) , Thermo Fisher Scientific , Cat# PA5-14149; RRID: AB_2231168.

Techniques: Recombinant, Membrane, Modification, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis, Gene Expression, Software, Sterility, Microscopy, Spectrophotometry, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Schematic diagram of the 4 DIV CNP differentiation protocol. b , Representative immunostaining images of H9 and GBX2 -/- CNPs at 4 DIV showing OTX2 - /CDX2 + cells. A few 4X CNPs were positive for OTX2, but no cells were positive for CDX2. Scale bars, 20 µm. c-d , qPCR analysis of OTX2 (c) and CDX2 (d) expression in H9, GBX2 -/- and 4X CNPs at 4 DIV. The data are presented as the mean ± SD; n= 3. One-way ANOVA showed statistical significance, and then an unpaired t-test comparing two groups was performed. *P< 0.05; **P< 0.01. e , Heatmap of the expression of pluripotent and neural genes representing the anterior, midbrain, hindbrain and spinal cord regions in H9, GBX2 -/- and 4X CNPs at 4 DIV. f , The top 10 downregulated (blue) and upregulated (red) genes (and additional selected genes in bold) between cultured 4X and H9 cells, cultured GBX2 -/- and H9 cells and cultured 4X and GBX2 -/- cells at 4 DIV. The threshold bar (white line) indicates a fold change of ±2. g , Schematic diagram of the 11 DIV CNP differentiation protocol. h , RNA expression analysis of the midbrain genes (orange) OTX2, EN1 and PAX8 ; the hindbrain genes (gray) MAFB, EGR2, HOXA2, HOXB1, HOXA3, HOXB2 , and HOXA4 ; and the spinal cord genes (purple) HOXB8 and HOXC10 . The data are presented as the mean ± SD; n= 3. One-way ANOVA followed by Tukey’s multiple comparisons test. *P< 0.05; **P< 0.01. i , Representative immunohistochemical analysis of OTX2/EN1 double-positive cells among 11 DIV 4X cells. No OTX2/EN1 double-positive cells were detected among H9 cells. Scale bars, 10 µm. For (b) and (i), DAPI was used as a nuclear stain. Caudal neural progenitor: CNP.

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Immunostaining, Expressing, Cell Culture, RNA Expression, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Schematic diagram of the 11 DIV CNP differentiation protocol using different concentrations of GSK3i ranging from 0.7 µM to 3 µM. b , Flow cytometry analysis of the percentage of OTX2-positive cells among H9, GBX2 -/- and 4X CNPs at 11 DIV. The data are presented as the mean ± SD; n= 3. Two-way ANOVA followed by Tukey’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. **P< 0.01. c , Schematic diagram of the 16 DIV caudal midbrain differentiation protocol using different concentrations of GSK3i ranging from 0.5 µM to 1 µM. d , Quantification of OTX2-positive cells among H9 and 4X cells at 16 DIV after administration of GSK3i at concentrations ranging from 0.5 µM to 1 µM. The data are presented as the mean ± SD; n= 4-8. Two-way ANOVA followed by Sidak’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. **P< 0.01. e , Expression of OTX2, EN1, LMX1A, CNPY1 , and HOXA2 in H9 and 4X cells treated with a range of GSK3i concentrations at 16 DIV. The data are presented as the mean ± SD., n= 3. Two-way ANOVA followed by Sidak’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. *P< 0.05; **P< 0.01. f , Representative immunohistochemical analysis of OTX2, EN1, FOXA2, and LMX1A expression in H9 and 4X cells treated with GSK3i at concentrations of 0.6 µM and 1 µM on 16 DIV. DAPI was used as a nuclear stain. Scale bars, 50 µm. Caudal neural progenitor: CNP.

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Flow Cytometry, Concentration Assay, Expressing, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , UMAP of cultured 4X and H9 cells at 16 DIV and a graph of the percentage of cells that each cell line contributed to each cluster (n = 10 4X cell spheres; n = 10 H9 cell spheres; total of 4,682 cells). b , Heatmap of the expression of selected genes illustrating the identity of the clusters. c, Percentage of 4X and H9 cells expressing OTX2 and EN1 . d , Feature plot of the contribution of each cell line to each cluster and feature plot of gene expression levels of OTX2, EN1, LMX1A, FOXA2, FGF8, HOXA/B family members and STMN2 . e , UMAP of cultured 4X and H9 cells at 62 DIV and a graph of the percentage of cells that each cell line contributed to each cluster (n = 4 4X cell cultures; n = 4 H9 cell cultures; total of 6,804 cells). f , Heatmap of the expression of selected genes illustrating the identity of the clusters. g , Feature plot of the contribution of each cell line to each cluster and feature plot of the gene expression levels of STMN2, DCX, TH, LMX1A, EN1, SHH and COL3A1 .

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Cell Culture, Expressing

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Overview of the in vivo study. Unilateral 6-OHDA-induced MFB lesions were generated (week -4) and confirmed 3 weeks later by the cylinder and amphetamine-induced rotation tests. The animals were subdivided into 3 groups with similar average scores on the rotation test. Four weeks after lesioning (week 0), two of these subgroups were transplanted with 250,000 cells (H9 or 4X cells), and the third group did not undergo transplantation (6-OHDA lesion group). The rotation test was repeated at weeks 8 and 18 posttransplant, and the cylinder test was repeated at week 18. The animals were killed at week 19 posttransplantation (23 week after lesioning) for histological analysis. b , Amphetamine-induced rotational asymmetry. Two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; time: F(1.689, 35.46) =19.50, P<0.0001; treatment: F (2, 21) = 15.23 P< 0.0001. ** P< 0.01 and ****P< 0.0001 vs. the 4X cell-transplanted group at the same time point. §§ P< 0.01 and §§§§P<0.0001 vs. the same group at week -1. c , The use of each forelimb (contra or ipsi) and both forelimbs in the cylinder test was analyzed by two-way repeated measures ANOVA followed by Sidak’s multiple comparison test with time and group as variables. Time x group: both: F (2, 22) = 5.785, P=0.009; ipsi: F (2, 22) = 8.800, P=0.001; contra: F (2, 22) = 4.642, P=0.021. *P<0.05 and **P<0.01 vs. the same group at -1 week. $P<0.05 and $$P<0.01 vs. the 6-OHDA lesion group at the same time point. £P<0.05 and ££P<0.01 vs. the H9 cell-transplanted group at the same time point. The data in (b) and (c) are presented as the mean ± SEM. n= 7 rats in the 6-OHDA lesion group, n = 9 rats in the 4X cell-transplanted group, and n=8 rats in the H9 cell-transplanted group). d , Representative photos of coronal sections from all three groups immunostained for TH. Higher magnification images of the areas in the frame are shown on the right. Scale bars, 50 µm for all three photos in the column. The graphs on the right show (e) the estimated numbers of TH-positive cells in the grafts, (f) the yield of TH-positive neurons per 100,000 grafted cells and (g) the volume of the TH-positive graft (see Methods for details). h-i , Representative photomicrographs showing HNA-positive and TH-positive cells within H9 cell (h) and 4X cell (i) grafts. The squares in h-i and h’-i’ indicate the magnified areas shown in h’-i’ and h’’-i’’ , respectively. DAPI was used as a nuclear stain. Scale bars, 200 μm (h-i) and 50 μm ( h’-i’) . j-n , Representative immunofluorescence images of cells double-positive for TH/FOXA2 (j) , TH/LMX1A (k) , TH/EN1 (l) , TH/GIRK2 (m) and TH/CALB1 (n) within 4X cell grafts. (j’-n’) High-power images of j-n highlighting the graft composition. Scale bar, 50 μm. o , Quantitative analysis of the immunofluorescence data in m and n, showing the percentages of GIRK2/TH and CALB1/TH double-positive cells within 4X cell grafts. The data are presented as the mean percentage ± SD (n=9 rats).

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: In Vivo, Generated, Transplantation Assay, Staining, Immunofluorescence