tb green premix extaqtm ii  (TaKaRa)

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet: TLX3 regulates proliferation of granule neuron progenitors in cerebellum (A) Immunofluorescence staining of TLX3 and Pax6 (marker of cerebellar granule neurons) in the sagittal section of cerebellum at PN7. Scale bar, 200μm. (B) Immunofluorescence staining of TLX3 and Ki67 (proliferation marker) in the sagittal section of cerebellum at PN7. Scale bar, 200μm. (C) Heatmap showing the expression of Tlx3 in various clusters of cerebellum identified from scRNA-seq published database beginning from E10 to PN10. (D) Cartoon illustrating the generation of Tlx3 conditional knockout mice (Tlx3 fl/fl ; Atoh1 Cre) . (E) Bar graph depicts real-time PCR analysis of Tlx3 confirming deletion of exon 2 at different stages of development in Tlx3 cKO. (F and G) Immunofluorescence staining of BrdU (12 h BrdU pulsing) and Pax6 (marker of cerebellar granule neurons) in the sagittal section of E16 control cerebellum ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout (Tlx3 fl/fl ; Atoh1 Cre +/− ) . Scale bar, 100μm. (f’ and g’) Enlarged image of the boxed region from (F and G), respectively. (H and I) Immunofluorescence staining of BrdU (12 h BrdU pulsing) and Pax6 (marker of cerebellar granule neurons) in the sagittal section of PN7 control cerebellum ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout ( Tlx3 fl/fl ; Atoh1 Cre +/− ). Scale bar, 200μm. (h’, h’’, i’, and i’’) Enlarged view of the boxed region from figures H and I respectively. (J) Quantification of the number of BrdU +ive and Pax6 +ive granule cells in the EGL layer of cerebellar cortex at E16 ( n = 9 for both genotypes). (K) Quantification of the number of BrdU +ive and Pax6 +ive granule cells in the EGL layer of cerebellar cortex at PN7 ( n = 9 for both genotypes). Data are represented as mean ± SD. ∗ p < 0.05, ∗∗∗ p < 0.001 (Unpaired-t test). See also Figures S1–S6 .

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Control

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet: TLX3 is a crucial determinant for patterning along the anterior-posterior axis of developing cerebellum (A–H) DAPI staining of sagittal sections of the cerebellum including the vermis of control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout (Tlx3 fl/fl ; Atoh1 Cre +/− ) from various developmental time points such as E16, E18, PN7, and PN21. Scale bar, 100μm. (c’and d’) Enlarged images of E18. (C and D) DAPI staining from the primary fissure. Arrow and arrowheads indicate changes in the cerebellar lobe pattern. (I) Appearance of the dorsal view of brain isolated from control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout mice (Tlx3 fl/fl ; Atoh1 Cre +/− ) at PN21 stage. Arrows indicate the changes in the cerebellar lobule arrangement. (J) Weight of the whole brain without olfactory bulb and cerebellum from control ( Tlx3 fl/fl ;Atoh1 Cre −/− ) and Tlx3 conditional knockout mice (Tlx3 fl/fl ; Atoh1 Cre +/− ) at PN21 stage ( n = 3 for both genotypes). (K) Quantitative measurement of cerebellar cross-sectional area of control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout mice (Tlx3 fl/fl ; Atoh1 Cre +/− ) at E16, E18, PN7 and PN21 stages ( n = 9 for both genotypes). Images (A–H) were generated through stitching multiple images using Photoshop software. Nuclei were stained with DAPI. Data are represented as mean ± SD. ∗∗∗ p < 0.001 (Unpaired-t test).

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Staining, Control, Knock-Out, Isolation, Generated, Software

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet: scRNA Sequencing profiling of PN4 cerebellum reveals cell type proportion changes in Tlx3 cKOs (A) UMAP representation of the scRNA-seq of PN4 posterior cerebellum from control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− ) . Dots represents the individual cells and colors indicate the cell clusters. (B) Expression of Tlx3 , Atoh1 , Cre and specific marker genes for each cluster identification from control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout (Tlx3 fl/fl ; Atoh1 Cre +/− ). Dot sizes represent the percentage of cells expressing a gene of interest. Colors show cell clusters, cell cycle phase and genotype of control and cKO. (C) Cell type proportion analysis of each clusters of control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout (Tlx3 fl/fl ; Atoh1 Cre +/− ). ∗ p < 0.05, ∗∗∗ p < 0.001(FisherTest). (D and E) Immunofluorescence staining of Pax6 (marker of cerebellar granule neurons) in the sagittal section of cerebellum at PN7 stage. Scale bar, 100μm. (F and G) Immunofluorescence staining of calbindin (marker of Purkinje neurons) in the sagittal section of cerebellum at PN7. Scale bar,100μm. (H) Quantification of the number of Pax6 +ive granule cells in the EGL layer of cerebellar ( n = 9 for both genotypes, Unpaired T-Test). (I) Quantification of the number of calbindin +ive Purkinje cells in the cerebellar cortex ( n = 9 for both genotypes, Unpaired T-Test). (J and K) Volcano plots showing the differential gene expression (DGE) between control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and cKO (Tlx3 fl/fl ; Atoh1 Cre +/− ) in clusters of granule neuron progenitors (GNP) and granule neurons (GN). Dots represent the genes. Dashed lines depicts the adjusted p -value down to ∗∗∗ p < 0.0001. (L) Gene Ontology overrepresentation analysis of up regulated (Control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) vs. cKO (Tlx3 fl/fl ; Atoh1 Cre +/− ) genes in GNP and GN clusters. Dot size represents the fold enrichment of genes referring to respective GO-term. (H and I) Data are represented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001(Un-paired t test). The number of animals is represented by “n”. See also Figures S7–S13 .

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Sequencing, Control, Expressing, Marker, Knock-Out, Immunofluorescence, Staining, Gene Expression

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet: TLX3 directly regulates the expression of anti-proliferative genes and its dysfunction leads to the decreased differentiation of granule neurons (A) Venn diagram showing the overlap of genes between TLX3 direct targets (2173 genes) and differentially expressed genes from control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and cKO (Tlx3 fl/fl ; Atoh1 Cre +/− ) in clusters GNP (37) and GN (123). (B) Violin plot representing the expression of anti-proliferative ( Cadm1, Cdk2ap1, Tgfb2 ) and microtubule stabilizing genes (Bbip1 and Insc) in each clusters identified from scRNA seq of control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and cKO (Tlx3 fl/fl ; Atoh1 Cre +/− ) . (C) UMAP plot representing the anti-proliferative genes ( Cadm1, Cdk2ap1, Tgfb2 ) obtained from overlap analyses. (D and E) Immunofluorescence staining of BrdU +ive and Pax6 +ive granule cells in the migrating EGL layer of cerebellar cortex at PN14 after 7 days of BrdU pulsing. Scale bar, 50μm. (F and G) Immunofluorescence staining of BrdU +ive and NeuN +ive granule cells in the differentiated IGL layer of cerebellar cortex at PN14 after 7 days of BrdU pulsing. Scale bar, 50μm. (H) Quantification of the number of BrdU +ive and Pax6 +ive granule cells in the migrating EGL layer of cerebellar cortex at PN14 corresponding to D and E. (I) Quantification of the number of BrdU +ive and NeuN +ive granule cells in the differentiated IGL layer of cerebellar cortex at PN14 corresponding to F and G. ( n = 9 for both genotypes). (H and I) Data are represented as mean ± SD.∗∗ p < 0.01, ns = 0.198(Un-paired t test). The number of animals is represented by “n”. See also Figures S14 and .

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Expressing, Control, Immunofluorescence, Staining

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet: TLX3 is crucial for forming the correct synaptic connection architecture of the cerebellum (A–F) Immunofluorescence staining of parvalbumin (marker for Purkinje neurons) and VGLUT1 (marker of parallel fiber extension of granule neurons) in the sagittal section of cerebellum at PN7 and PN21 in control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− ) . Scale bar, 20μm (A and B), 50 μm (C–F). (G) Quantification of the number of VGLUT1 puncta on to each Purkinje cell body of images A–F. ( n = 9 for both genotypes). (H–M) Immunofluorescence staining of parvalbumin (marker for Purkinje neurons) and VGLUT2 (marker of climbing fiber) in the sagittal section of cerebellum at PN7 and PN21 in control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− ) . Scale bar, 20μm (H and I), 50 μm (J-M). (a’,b’, e’ f’, h’, and i’) Enlarged images of A, B, E, F, H, and I, respectively. (N) Quantification of the number of VGLUT2 puncti on to each Purkinje cell body of images H–M. ( n = 9 for both genotypes). (O) Violin plot showing the percentage of climbing fiber extensions into each Purkinje neurons at PN21 in control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockout (Tlx3 fl/fl ; Atoh1 Cre +/− ) . ( n = 9 for both genotypes). (G, N, and O) Data are represented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001(Un-paired t test). The number of animals is represented by “n”. Arrow indicate puncti. See also Figure S16 .

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Immunofluorescence, Staining, Marker, Control, Knock-Out

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet: TLX3 deficient mice exhibit defects in motor and social function (A) Latency to fall in the two limb wire hang test for control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 12) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 9). (B) Latency to fall in the four limb wire hang test for control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 12) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 14). (C) Latency to fall in the rotarod test for control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 8) and Tlx3 conditional knockout (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 8). (D) Total distance traveled by control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 11) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 9) in the open maze chamber. (E) Time spent in the open maze central zone by control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 12) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 10). (F) Time spent in the open arm of elevated plus maze by control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 13) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 13). (G) Interaction time of control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 6) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 6) with familiar and novel object in the Novel object recognition test. (H) Number of marbles buried by control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 13) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 15) in the marble burying test. (I) Social preference test results of control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 8) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 7) which shows the interaction time of each mouse with social stimuli and empty cup. (J) Social novelty test results of control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 8) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 7) that shows the interaction time taken by each mouse with familiar social stimuli and novel social stimuli. (K) Rate of calling of control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 6) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 5) upon introduction of intruder mice in the resident –intruder test and ultra-sonic vocalization recording. (L) Peak frequency of the calls produced by control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 6) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 5) during resident –intruder test and ultra-sonic vocalization recording. (M) Latency for initiating the first ultrasonic call when intruder is introduced with the control ( Tlx3 fl/fl ; Atoh1 Cre −/− , n = 6) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− , n = 5). (A–M) Data are represented as mean ± SD. (A–F, H, and M) Data are represented as box and whiskers plot (Mann-Whitney test Unpaired(U)-Tests in A–F and H, Mann-Whitney test U-Tests and Ordinary one-way ANOVA for group analysis in H–J and Unpaired-t test in K–M). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). The number of animals is represented by “n”. See also Figures S17–S19 .

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Control, Knock-Out, Produced, MANN-WHITNEY

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet: Deletion of Tlx3 shows altered expression of the ASD Candidate genes in GNPs of Cerebellum (A) Dot plot visualizing the genes obtained from overlap analysis of differentially expressed genes from scRNA Seq of GNP and GN clusters with SFARI ASD database. Size of the dot is determined by the gene score. (B) Violin plot representing the expression of overlapping genes obtained from overlap analysis of differentially expressed genes from scRNA Seq of GNP and GN clusters of control ( Tlx3 fl/fl ; Atoh1 Cre −/− ) and Tlx3 conditional knockouts (Tlx3 fl/fl ; Atoh1 Cre +/− ) with SFARI ASD database. Colors show genotype of control and KO. (C) Dot plot depicting the distribution of missense and structural variations of the TLX3 gene in the global population, determined by the analysis of minor allele frequencies. (D) Pie diagram showing the percentage of TLX3 variations in association with various pathological conditions curated from published literature and ClinVar database.

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Expressing, Control

Journal: iScience

Article Title: TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism

doi: 10.1016/j.isci.2024.111260

Figure Lengend Snippet:

Article Snippet: Atoh1 Cre genotyping primer (Transgene) F-CCGGCAGAGTTTACAGAAGC R-ATGTTTAGCTGGCCCAAATG , The Jackson Laboratory, USA , N/A.

Techniques: Recombinant, Electron Microscopy, DNA Extraction, Random Hexamer, Reverse Transcription, RNA Extraction, In Situ, Positive Control, Software, Control, Sequencing