Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells
- Laboratory of Developmental Neurobiology, Rockefeller University, United States
- Bioinformatics Resource Center, Rockefeller University, United States
Figures
Figure 1 with 3 supplements
Derivation of the human ATOH1 lineage.
(A) EN2 expression (log10 fold change of no CHIR99021) in dual SMAD+FGF2-treated human pluripotent stem cells (hPSCs) in the absence and presence of CHIR99021 by RT-qPCR at day in vitro (DIV) 11. (B)…
Figure 1—figure supplement 1
Derivation and characterization of the human ATOH1 lineage.
(A) Gene expression by RT-PCR at days in vitro (DIV) 4, 7, and 11 comparing dual SMAD inhibition plus FGF2/insulin versus FGF2/CHIR99021 treatment. (B) Top: schematic showing 11 different time …
Figure 1—figure supplement 2
Characterization of ATOH1-EGFP and ATOH1-EGFP-L10a transgenic lines.
(A) Left: representative image of EGFP expression upon differentiation of the ATOH1-EGFP lines at day in vitro (DIV)16. Middle: normal karyotype detected. Right: bar chart showing the mean ± SD of …
Figure 1—figure supplement 3
Characterization of ATOH1-EGFP cells (until day in vitro [DIV] 28).
(A) Top panel: Calretinin and NeuN expression in the cerebellar nuclei at P0 in mice. Lower panel: Calretinin, NeuN, and ATOH1-EGFP expression in human pluripotent stem cell (hPSC) cultures at …
Figure 2 with 1 supplement
Human granule cell (GC) differentiation from human pluripotent stem cells (hPSCs).
(A) TAG1 expression at day in vitro [DIV]18 (left) and DIV28 (right). (B) Schematic of the sorting strategy of TAG1+ cells by magnetic-activated cell sorting (MACS) at DIV28 and co-culture with …
Figure 2—figure supplement 1
Characterization of TAG1-negative fraction at day in vitro (DIV)28 + 20.
(A) NEUROD1 expression and (B) NeuN and Calretinin expression in the TAG1-negative fraction after magnetic-activated cell sorting at DIV28 followed by differentiation of cells for another 20 days …
Figure 3 with 1 supplement
Human postmitotic granule cells (GCs) undergo glial-guided neuronal migration and integrate into the mouse cerebellum upon transplantation.
(A) Left: schematic outlining transplantation of MACsorted (day in vitro [DIV]28–32) TAG1+ human cells into the early postnatal mouse cerebellum. Images show three representative coronal sections of …
Figure 3—figure supplement 1
Integration of human pluripotent stem cell-granule cells (hPSC-GCs) into the mouse cerebellum upon transplantation.
Example of a coronal section of a mouse cerebellum showing human cells (blue), some of which integrated into the internal granule cell layer (IGL) (arrowheads) below the Purkinje cell layer marked …
Figure 4 with 2 supplements
The human pluripotent stem cell (hPSC)-derived ATOH1 lineage resembles the human cerebellum in the second trimester by translational profiling.
(A) Volcano plot of log2 fold change global gene expression in ATOH1-TRAP IPs versus input. Key granule cell (GC) genes are highlighted by red dots (Figure 4—source data 1). The fully differentiated …
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Figure 4—source data 1
DESeq2 analysis of ATOH1-EGFP-L10a TRAP IP versus input.
- https://cdn.elifesciences.org/articles/67074/elife-67074-fig4-data1-v2.xlsx
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Figure 4—source data 2
Comparison of ATOH1-EGFP-L10a TRAP IP to human developmental data from PsychEncode.
- https://cdn.elifesciences.org/articles/67074/elife-67074-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Heatmaps of key developmental signaling pathways in the hPSC-ATOH1 lineage.
(A) RNA sequencing of immunoprecipitated mRNAs from an ATOH1-EGFP-L10a hPSC TRAP line differentiated until day in vitro [DIV]28 shows enrichment of ATOH1 reads in the ATOH1-TRAP IPs compared to the …
Figure 4—figure supplement 2
Comparison of ATOH1-EGFP-L10a TRAP against single-cell RNA-seq data from the developing mouse cerebellum.
Dot plot representation of the cumulative enrichment (by p-value) of the ATOH1-EGFP-L10a data against glutamatergic, GABAergic, and non-neuronal cell clusters identified by Wizeman et al., 2019.
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Figure 4—figure supplement 2—source data 1
Comparison of ATOH1-EGFP-L10a TRAP IP to scRNA-seq from Wizeman et al., 2019, See Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/67074/elife-67074-fig4-figsupp2-data1-v2.xlsx
Figure 5 with 1 supplement
Temporal shift in the expression of transcriptional regulators in the human external granule cell layer (EGL) compared to mouse.
(A) Left: bar chart showing the mean ± 1 SD normalized expression of transcriptional regulators in ATOH1-TRAP IPs at day in vitro (DIV) 28 by RNA-seq. Right: the expression of PAX6 (GCP marker), and …
Figure 5—figure supplement 1
Marker expression in the developing human cerebellum versus mouse.
(A) Box plot showing the percentages of NEUROD1+ nuclei per total nuclei (Dapi) along the length of the pial surface in anterior, midline, and posterior regions of the developing cerebellum …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | RUES2 | Human embryonic stem cell line | NIH registration number: NIHhESC-09-0013 | |
| Cell line (human) | H9 (WA09) | Human embryonic stem cell line | NIH registration number: NIHhESC-10-0062 | |
| Cell line (human) | ATOH1-EGFP | Human embryonic stem cell line | This study (RUES2 line) | |
| Cell line (human) | ATOH1-EGFP-L10a | Human embryonic stem cell line | This study (RUES2) | |
| Recombinant DNA reagent | pPS-EF1α-GFP-RFP | System Biosciences | LV603PA-1 | Lentiviral vector |
| Recombinant DNA reagent | J2XnGFP | Dr. Jane Johnson | GFP plasmid | |
| Recombinant DNA reagent | pSIN-hATOH1 enhancer- hβ-globin-nlsEGFP-bGH polyA-hPGK-Puromycin | This study | ATOH1-EGFP lentiviral construct | |
| Recombinant DNA reagent | pSIN-hATOH1 enhancer- hβ-globin-nlsEGFP-bGH polyA-hPGK-Puromycin | This study | ATOH1-EGFP- L10a lentiviral construct | |
| Biological sample (human) | Human fetal cerebellum (17 PCW) | Human Developmental Biology Resource | http://www.hdbr.org/ | |
| Biological sample (mouse) | C57Bl/6J mice | Jackson Laboratory | Embryos and pups obtained from times matings | |
| Antibody | Calretinin (rabbit polyclonal) | Swant | 7699/4 | (1:1000) |
| Antibody | EN2 (C19) (goat polyclonal) | Santa Cruz | SC-8111 | (1:50) |
| Antibody | GFAP (chicken polyclonal) | EnCor | CPCA-GFAP | (1:1500) |
| Antibody | GFP (rabbit polyclonal) | Invitrogen | A-111122 | (1:500) |
| Antibody | GFP (chicken polyclonal) | Aves labs | GFP-1020 | (1:1000) |
| Antibody | HuNu (anti-human nuclei) (mouse monoclonal) | Millipore | MAB1281 | (1:100) |
| Antibody | Ki67 (rabbit monoclonal) | Vector Laboratories | VP-RM04 | (1:100) |
| Antibody | Ki67 (rabbit polyclonal) | EnCor | RPCA-Ki67 | (1:1000) |
| Antibody | Map2 (chicken polyclonal) | Abcam | ab5392 | (1:1000) |
| Antibody | NeuN (mouse monoclonal) | Millipore | MAB377 | (1:100) |
| Antibody | NeuroD1 (mouse monoclonal) | BD Pharmingen | 563000 | (1:300) |
| Antibody | Pax6 (rabbit polyclonal) | BioLegend | 901301 | (1:300) |
| Antibody | TAG1 (mouse monoclonal) | Tom Jessell | (1:2) | |
| Antibody | Synaptophysin (mouse monoclonal) | Millipore | MAB329 | (1:500) |
| Antibody | VGLuT1 (mouse monoclonal) | Millipore | MAB5502 | (1:100) |
| Antibody | SOX2 (rabbit monoclonal) | Cell Signaling | 3579 | (1:200) |
| Antibody | GluR-d2 (goat polyclonal) | Santa Cruz | Sc-26118 | (1:100) |
| Antibody | Anti-goat Alexa Fluor 633 (donkey polyclonal) | Invitrogen | A21082 | (1:300) |
| Antibody | Anti-rabbit Alexa Fluor 555 (donkey polyclonal) | Invitrogen | A-31572 | (1:300) |
| Antibody | Anti-mouse IgM Alexa Fluor 488 (goat polyclonal) | Invitrogen | A-21042 | (1:300) |
| Antibody | Anti-mouse Alexa Fluor 555 (donkey polyclonal) | Invitrogen | A-31570 | (1:300) |
| Antibody | Anti-chicken IgY 488 (donkey polyclonal) | Jackson ImmunoResearch | 703-545-155 | (1:300) |
| Antibody | Anti-chicken IgY Cy3 (donkey polyclonal) | Jackson ImmunoResearch | 703-165-155 | (1:300) |
| Antibody | Anti-rabbit Alexa Fluor 647 (donkey polyclonal) | Invitrogen | A-31573 | (1:300) |
| Antibody | Anti-mouse Alexa Fluor 647 (donkey polyclonal) | Invitrogen | A-31571 | (1:300) |
| Antibody | Anti-mouse Alexa Fluor 488 (donkey polyclonal) | Invitrogen | A-21202 | (1:300) |
| Antibody | Anti-mouse Fluor 405 (donkey polyclonal) | Abcam | Ab175658 | (1:300) |
| Sequence-based reagent | ATOH1 | This paper | PCR primer | Forward 5′-GCGCA AAAGAATTT GTCTCC-3′ |
| Sequence-based reagent | ATOH1 | This paper | PCR primer | Reverse 5′-GCG AAGTTTTGCTG TTTTCC-3′ |
| Sequence-based reagent | ID4 | This paper | PCR primer | Forward 5′-GC TCACTGCGCT CAACACC-3′ |
| Sequence-based reagent | ID4 | This paper | PCR primer | Reverse 5′-GAA TGCTGTCGCC CTGCTTG-3′ |
| Sequence-based reagent | EN2 | This paper | PCR primer | Forward 5′- GG CGTGGGTCTA CTGTACG-3′ |
| Sequence-based reagent | EN2 | This paper | PCR primer | Reverse 5′- TACCTGTTG GTCTGGAA CTCG-3′ |
| Sequence-based reagent | PAX6 | This paper | PCR primer | Forward 5′-TCA CCATGGCAA ATAACCTG-3′ |
| Sequence-based reagent | PAX6 | This paper | PCR primer | Reverse 5′-CA GCATGCAGG AGTATGAGG-3′ |
| Sequence-based reagent | NEUROD1 | This paper | PCR primer | Forward 5′-GGACGA GGAGCAC GAGGCAG ACAAGAA-3′ |
| Sequence-based reagent | NEUROD1 | This paper | PCR primer | Reverse 5′- TTCCTCA GTGAGTCC TCCTCTG CGTTCA-3′ |
| Sequence-based reagent | PCP2 | This paper | PCR primer | Forward 5′- GACC AGGAGGG CTTCTTCAATCT-3′ |
| Sequence-based reagent | PCP2 | This paper | PCR primer | Reverse 5′- CATG TCCATGA GGCTGT CCATCT-3′ |
| Sequence-based reagent | OTX2 | This paper | PCR primer | Forward 5′-ACAA GTGGC CAATTCA CTCC-3′ |
| Sequence-based reagent | OTX2 | This paper | PCR primer | Reverse 5′-GAGG TGGACAA GGGATCTGA-3′ |
| Sequence-based reagent | MEIS2 | This paper | PCR primer | Forward 5′-CCAG GGGACT ACGTTTCTCA-3′ |
| Sequence-based reagent | MEIS2 | This paper | PCR primer | Reverse 5′-TAA CATTGT GGGGC TCTGTG-3′ |
| Sequence-based reagent | GBX2 | This paper | PCR primer | Forward 5′-GTTCC CGCCG TCGCTGATGAT-3′ |
| Sequence-based reagent | GBX2 | This paper | PCR primer | Reverse 5′-GCC GGTGTA GACGAA ATGGCCG-3′ |
| Sequence-based reagent | HOXA2 | This paper | PCR primer | Forward 5-CGT CGCTC GCTGA GTGCCTG-3′ |
| Sequence-based reagent | HOXA2 | This paper | PCR primer | Reverse 5′-TGTC GAGTGTG AAAGCG TCGAGG-3′ |
| Sequence-based reagent | LHX2 | This paper | PCR primer | Forward 5′- GGTCCTC CAGGTCT GGTTC-3′ |
| Sequence-based reagent | LHX2 | This paper | PCR primer | Reverse 5′- TAAGAG GTTGCGC CTGAACT-3′ |
| Sequence-based reagent | LHX9 | This paper | PCR primer | Forward 5′- GCT GGGAGT GGACATCGTCA-3′ |
| Sequence-based reagent | LHX9 | This paper | PCR primer | Reverse 5′- CATG GTCCGGA GCTGGTGAT-3′ |
| Sequence-based reagent | β-ACTIN | This paper | PCR primer | Forward 5′-AAAC TGGAAC GGTGAAGG-3′ |
| Sequence-based reagent | β-ACTIN | This paper | PCR primer | Reverse 5′-AGA GAAGT GGGGTGGCTT-3′ |
| Sequence-based reagent | ATP5O | This paper | PCR primer | Forward 5′- cgcta tgccac agctcttta-3′ |
| Sequence-based reagent | ATP5O | This paper | PCR primer | Reverse 5′- atgg aacgcttc acataggg-3′ |
| Peptide, recombinant protein | Human bFGF | Invitrogen | Catalog # 13256-029 | |
| Peptide, recombinant protein | Human/mouse/ ratBDNF | PeproTech | Catalog # 450-02 | |
| Peptide, recombinant protein | Mouse BMP7 | R&D Systems | Catalog # 5666BP-010 | |
| Peptide, recombinant protein | Human recombinant insulin | Tocris | Catalog # 3435 | |
| Peptide, recombinant protein | Human BMP4 | R&D Systems | Catalog # 314BP-050 | |
| Peptide, recombinant protein | Human BMP6 | R&D Systems | Catalog # 507BP-020 | |
| Peptide, recombinant protein | Human/mouse FGF8b | R&D Systems | Catalog # 423-F8-025 | |
| Commercial assay or kit | RNeasy micro kit | QIAGEN | Catalog # 74004 | |
| Commercial assay or kit | RNeasy Plus mini kit | QIAGEN | Catalog # 74134 | |
| Commercial assay or kit | Transcription First Strand cDNA Synthesis Kit | Roche Life Sciences | Catalog # 04379012001 | |
| Commercial assay or kit | HotStarTaq PLUS DNA Polymerase kit | QIAGEN | Catalog # 203603 | |
| Commercial assay or kit | Click-iT EdU Cell Proliferation Kit for Imaging | Invitrogen | Catalog # C10338 | |
| Commercial assay or kit | SMART-Seq v4 Ultra Low Input RNA Kit | TaKaRa Bio | Catalog # 634888 | |
| Commercial assay or kit | Nextera XT DNA library preparation kit | Illumina | Catalog # FC-131-1024 | |
| Commercial assay or kit | RNA 6000 Pico Kit | Agilent | Catalog # 5067-1513 | |
| Commercial assay or kit | In-fusion HD cloning plus (Clontech) | TaKaRa Bio | Catalog # 638909 | |
| Commercial assay or kit | Anti-mouse IgM microbeads | Miltenyi Biotec | Catalog # 130-047-302 | |
| Commercial assay or kit | MS columns | Miltenyi Biotec | Catalog # 130-042-201 | |
| Chemical compound, drug | ROCK-inhibitor Y-27632 | Abcam | Catalog # ab 120129 | |
| Chemical compound, drug | SB431542 | Tocris | Catalog # 1614 | |
| Chemical compound, drug | LDN-193189 | Stemgent/ Tocris | Catalog # 6053 | |
| Chemical compound, drug | CHIR99021 | Stemgent/ REPROCELL | Catalog # 04-0004-02 | |
| Software, algorithm | Primer3 | Open source | Primer3, RRID:SCR_003139 | |
| Software, algorithm | Salmon quantification software (version 0.8.2) | Open source | Salmon, RRID:SCR_017036 (Patro et al., 2017) | |
| Software, algorithm | R statistical software | Open source | R Project for Statistical Computing, RRID:SCR_001905 | |
| Software, algorithm | Tximport (version 1.8.0). | Open source | tximport, RRID:SCR_016752 (Love et al., 2016) | |
| Software, algorithm | DESeq2 (version 1.20.0) | Open source | DESeq2, RRID:SCR_015687 (Love et al., 2018) | |
| Software, algoritham Software, algorithm | rtracklayer package (version 1.40.6) | Open source | rtracklayer, RRID:SCR_021325 | |
| Software, algorithm | GSVA (version 1.34.0) | Open source(Hänzelmann et al., 2013) | GSVA, RRID:SCR_021058 | |
| Software, algorithm | Pheatmap R package (version 1.0.10) | Open source | ncv | pheatmap, RRID:SCR_016418 |
| Software, algorithm | topGO Bioconductor package | Open source | topGO, RRID:SCR_014798 | |
| Software, algorithm | GOseq Bioconductor package | Open source (Young et al., 2010) | Goseq, RRID:SCR_017052 | |
| Software, algorithm | ImageJ (version 2.1.0/1.53c) | Open source, NIH | ImageJ, RRID:SCR_003070 | |
| Software, algorithm | SPSS software | IBM | ||
| Software, algorithm | BD FACSDiva 8.0.1 software | BD Biosciences | ||
| Software, algorithm | ZEN imaging software | Zeiss |
Table 1
Table of primers.
| Gene | Forward primer | Reverse primer | °C |
|---|---|---|---|
| ATOH1 | 5′-GCGCAAAAGAATTTGTCTCC-3′ | 5′-GCGAAGTTTTGCTGTTTTCC-3′ | 60 |
| ID4 | 5′-GCTCACTGCGCTCAACACC-3′ | 5′-GAATGCTGTCGCCCTGCTTG-3′ | 60 |
| EN2 | 5′-GGCGTGGGTCTACTGTACG-3′ | 5′-TACCTGTTGGTCTGGAACTCG-3′ | 59 |
| PAX6 | 5′-TCACCATGGCAAATAACCTG-3′ | 5′-CAGCATGCAGGAGTATGAGG-3′ | 60 |
| NEUROD1 | 5′-GGACGAGGAGCACGAGGCAGACAAGAA-3′ | 5′-TTCCTCAGTGAGTCCTCCTCTGCGTTCA-3′ | 56 |
| PCP2 | 5′-GACCAGGAGGGCTTCTTCAATCT –3′ | 5′-CATGTCCATGAGGCTGTCCATCT-3′ | 56 |
| OTX2 | 5′-ACAAGTGGCCAATTCACTCC-3′ | 5′-GAGGTGGACAAGGGATCTGA-3′ | 60 |
| MEIS2 | 5′-CCAGGGGACTACGTTTCTCA-3′ | 5′-TAACATTGTGGGGCTCTGTG-3′ | 50 |
| GBX2 | 5′-GTTCCCGCCGTCGCTGATGAT-3′ | 5′-GCCGGTGTAGACGAAATGGCCG-3′ | 60 |
| HOXA2 | 5-CGTCGCTCGCTGAGTGCCTG-3′ | 5′-TGTCGAGTGTGAAAGCGTCGAGG-3′ | 60 |
| LHX2 | 5′- GGTCCTCCAGGTCTGGTTC-3′ | 5′-TAAGAGGTTGCGCCTGAACT-3′ | 60 |
| LHX9 | 5′- GCTGGGAGTGGACATCGTCA-3′ | 5′-CATGGTCCGGAGCTGGTGAT-3′ | 60 |
| β-ACTIN | 5′-AAACTGGAACGGTGAAGG-3′ | 5′-AGAGAAGTGGGGTGGCTT-3′ | 59 |
| ATP5O | 5′-cgctatgccacagctcttta-3′ | 5′-atggaacgcttcacataggg-3′ | 60 |
Table 2
Table of antibodies.
| Antibody | Species | Source | Dilution | Catalog # |
|---|---|---|---|---|
| Calretinin | Rabbit | Swant | 1:1000 | 7699/4 |
| EN2 (C19) | Goat | Santa Cruz | 1:50 | SC-8111 |
| GFAP | Chicken | EnCor Biotechnology | 1:1500 | CPCA-GFAP |
| GFP | Rabbit | Invitrogen | 1:500 | A-111122 |
| GFP | Chicken | Aves labs | 1:1000 | GFP-1020 |
| HuNu (anti human nuclei) | Mouse | Millipore | 1:100 | MAB1281 |
| Ki67 | Rabbit | Vector Laboratories | 1:100 | VP-RM04 |
| Ki67 | Rabbit | EnCor | 1:1000 | RPCA-Ki67 |
| MAP2 | Chicken | Abcam | 1:1000 | ab5392 |
| NEUN | Mouse | Millipore | 1:100 | MAB377 |
| NEUROD1 | Mouse | BD Pharmingen | 1:300 | 563000 |
| PAX6 | Rabbit | BioLegend | 1:300 | 901301 |
| TAG1 | Mouse IgM | T.Jessell | 1:2 | N/A |
| Synaptophysin | Mouse IgM | Millipore | 1:500 | MAB329 |
| VGLUT1 | Mouse | Millipore | 1:100 | MAB5502 |
| SOX2 (D6D9) | Rabbit | Cell Signaling | 1:200 | 3579 |
| GluR-δ2 | Goat | Santa Cruz | 1:100 | Sc-26118 |
| Anti-goat Alexa Fluor 633 | Donkey | Invitrogen | 1:300 | A21082 |
| Anti-rabbit Alexa Fluor 555 | Donkey | Invitrogen | 1:300 | A-31572 |
| Anti-mouse IgM Alexa Fluor 488 | Goat | Invitrogen | 1:300 | A-21042 |
| Anti-mouse Alexa Fluor 555 | Donkey | Invitrogen | 1:300 | A-31570 |
| Anti-chicken IgY 488 | Donkey | Jackson ImmunoResearch | 1:300 | 703-545-155 |
| Anti-chicken IgY Cy3 | Jackson ImmunoResearch | 1:300 | 703-165-155 | |
| Anti-rabbit Alexa Fluor 647 | Donkey | Invitrogen | 1:300 | A-31573 |
| Anti-mouse 405 | Donkey | Abcam | 1:300 | Ab175658 |
| Anti-mouse Alexa Fluor 647 | Donkey | Invitrogen | 1:300 | A-31571 |
| Anti-mouse Alexa Fluor 488 | Donkey | Invitrogen | 1:300 | A-21202 |
Additional files
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Supplementary file 1
GO terms for TRAP-seq data.
- https://cdn.elifesciences.org/articles/67074/elife-67074-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67074/elife-67074-transrepform1-v2.docx
