Aberrant cortical development is driven by impaired cell cycle and translational control in a DDX3X syndrome model

  1. Mariah L Hoye
  2. Lorenzo Calviello
  3. Abigail J Poff
  4. Nna-Emeka Ejimogu
  5. Carly R Newman
  6. Maya D Montgomery
  7. Jianhong Ou
  8. Stephen N Floor
  9. Debra L Silver  Is a corresponding author
  1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, United States
  2. Centre for Functional Genomics, Human Technopole, Italy
  3. Centre for Computational Biology, Human Technopole, Italy
  4. Department of Cell Biology, Duke University Medical Center, United States
  5. Duke Regeneration Center, Duke University Medical Center, United States
  6. Department of Cell and Tissue Biology, UCSF, United States
  7. Helen Diller Family Comprehensive Cancer Center, United States
  8. Department of Neurobiology, Duke University Medical Center, United States
  9. Duke Institute for Brain Sciences, Duke University Medical Center, United States
9 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
Conditional knockout of Ddx3x in neural progenitors using Emx1-Cre leads to microcephaly in female mice.

(A) Schematic of DDX3X protein with human missense and nonsense mutations noted, along with helicase/RNA binding domains (red, blue). Nonsense mutations, highlighted in yellow, are predicted to act …

Figure 1—figure supplement 1
Ddx3x loss from neural progenitors, but not neurons leads to microcephaly and apoptosis.

(A) Representative western blot of indicated genotypes probed for DDX3X (top) and β-actin loading control (bottom). Densitometric quantification of bands is shown below each lane. These data also …

Figure 1—figure supplement 1—source data 1

Western blot anlaysis of DDX3X in embryonic brain samples.

https://cdn.elifesciences.org/articles/78203/elife-78203-fig1-figsupp1-data1-v1.zip
Ddx3x knockout is sexually dimorphic and Ddx3y phenocopies Ddx3x loss.

(A) RT-qPCR quantification of Ddx3x and Ddx3y mRNA levels in FACS-isolated Tdtomato + cells from cKO male E11.5 cortices. n=5–8 embryos/condition. (B) Schematic of Ddx3y CRISPR sgRNA electroporation …

Figure 3 with 1 supplement
Ddx3x depletion leads to more RGCs and mature IPs, and fewer excitatory neurons across laminar layers.

(A) Representative sections from E14.5 cortices stained with Sox2 (magenta) and Tbr2 (green) (control M and cKO M shown). (B, C) Quantification of density of Sox2+ (RGCs) (B) and Tbr2 +Sox2- (mature …

Figure 3—figure supplement 1
Ddx3x depletion leads to more progenitors and less neurons at E13.5, but does not affect cortical thickness or laminar position of neurons.

(A) Representative coronal sections from control and cHet F/cKO M at E13.5 immunostained with Sox2 (green), Tbr2 (magenta) and Hoechst (control M and cKO M shown). (B–E) Quantification of RGCs …

Figure 4 with 1 supplement
Ddx3x depletion impairs progenitor cell cycle exit and prolongs cell cycle duration.

(A) Representative sections stained with Ki67 (green) and EdU (magenta) from E14.5 control and cHet F/cKO M mice (control F and cHet F shown) pulsed with EdU at E13.5, and higher magnification …

Figure 4—figure supplement 1
Ddx3x depletion prolongs cell cycle duration in RGCs and immature IPs but does not affect mitosis duration.

(A) Representative coronal sections from control and cHet F/cKO M at E14.5 immunostained with Sox2 (green), Tbr2 (red), EdU (magenta) and Hoechst (control F and cHet F shown). (B) Quantification of …

Ddx3x-depleted progenitors exhibit prolonged cell division and more proliferative divisions at the expense of neurogenic divisions.

(A) Live imaging paradigm for monitoring cell fate. Figure 5A has been adapted from Figure 3A and E from Pilaz et al., 2016. (B) Live imaging DIC snapshots at indicated t=minutes or hours, and fixed …

Figure 6 with 2 supplements
Ribosome Profiling in embryonic brains uncovers the E11.5 translatome including DDX3X-dependent translation targets.

(A) Experimental paradigm for Ribo-seq and RNAseq of E11.5 cortices from control and cKO mice. n=3/sex/condition with four embryos pooled per n. (B) TE of transcripts enriched in RGCs, IPs, deep …

Figure 6—figure supplement 1
Quality Control Assessment of Ribosome Profiling in Ddx3x cKO mice.

(A) Representative denaturing urea gel of embryonic cortices treated with RNase I illustrating RPFs (red box). (B–D) RibosomeProfilingQC assessment of deep sequencing of cDNA libraries showing read …

Figure 6—figure supplement 1—source data 1

Quality Control Assessment of Ribosome Profiling.

https://cdn.elifesciences.org/articles/78203/elife-78203-fig6-figsupp1-data1-v1.zip
Figure 6—figure supplement 2
Polysome fractionation and RNA immunoprecipitations showing DDX3X targets in the cortex.

(A–C) RT-qPCR quantification of mRNA levels for Ribo-seq candidates in input samples, monosome and polysome fractions at E11.5 for Topbp1 (A) and Setd3 (B) and at E14.5 for Hax1 (C). (D) …

Figure 6—figure supplement 2—source data 1

RNA immunoprecipitations of targets by DDX3X.

https://cdn.elifesciences.org/articles/78203/elife-78203-fig6-figsupp2-data1-v1.zip
DDX3X-dependent translation target, Setd3, is required for neurogenesis.

(A) RT-qPCR quantification of Setd3 knockdown in N2A cells. n=4/condition with two independent trials. (B) Representative sections of E17.5 brains from mice electroporated at E14.5 with pCAG-GFP and …

Author response image 1
Representative 100um optical sagittal sections (50um on either side of the midsagittal plane) of brains of noted genotype generated using Imaris software.

Scale bar = 300mm.

Author response image 2
Representative images of female cortices electroporated with Ddx3y gRNA.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Ddx3xNAMGI:103064; NCBI Gene: 13,205
Gene (M. musculus)Ddx3yNAMGI:1349406; NCBI Gene: 26,900
Gene (M. musculus)Rcor2NAMGI:1859854; NCBI Gene: 104,383
Gene (M. musculus)Setd3NAMGI:1289184; NCBI Gene: 52,690
Gene (M. musculus)Topbp1NAMGI:1920018; NCBI Gene: 235,559
Genetic reagent (M. musculus)C57BL/6 JJackson LaboratoryJAX #000664; RRID:IMSR_JAX:000664
Genetic reagent (M. musculus)Emx1-CreJackson LaboratoryJAX #005628; RRID:IMSR_JAX:005628; MGI:2684610MGI symbol: Emx1 tm1(cre)Krj
Genetic reagent (M. musculus)Rosa26Ai14Jackson LaboratoryJAX #007914; RRID:IMSR_JAX:007914MGI symbol: Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze
Genetic reagent (M. musculus)Neurod6-Cre (NEX-Cre)PMID:17146780MGI:2668659MGI symbol: Neurod6 tm1(cre)Kan
Genetic reagent (M. musculus)Ddx3xlox/loxPMID:27179789MGI:5774968MGI symbol: Ddx3x tm1.1Lyou
Genetic reagent (M. musculus)Ddx3y sgRNAsthis paperNCBI gene: 26,900generated with Benchling for depleting Ddx3y; see Figure 2
Genetic reagent (M. musculus)smFISH probes for Ddx3xthis paperNCBI gene: 13,205generated with script from Tsanov et al., 2016 for monitoring Ddx3x RNA; see Figure 1
Genetic reagent (M. musculus)Scrambled siRNAsQiagenQiagen:1022076
Genetic reagent (M. musculus)Setd3 siRNASQiagenQiagen:1027416Gene ID: 52,690
Cell line (M. musculus, male)Neuro-2aATCCATCC:CCL-131; RRID:CVCL_0470
Strain, strain backgroundNEB 5-alpha Competent E. coli (High Efficiency)New England BiolabsNEB:C2987H
Sequence-based reagent (M. musculus)Rcor2 qPCR primersHarvard PrimerBank; PMID:22086960Harvard PrimerBank ID: 154147710 c2; Forward 5'-TGCTTCTGTGGCATAAACACG-3'; Reverse 5'-GGCTGGGAATCACCTTGTCAG-3'
Sequence-based reagent (M. musculus)Setd3 qPCR primersHarvard PrimerBank; PMID:22086960Harvard PrimerBank ID: 21312266a1; Forward 5'-AAATCAGGTACTGGGGCTACA-3'; Reverse 5'-GGCCCATTTCATTAGATCAGGGA-3'
Sequence-based reagent (M. musculus)Topbp1 qPCR primersHarvard PrimerBank; PMID:22086960Harvard PrimerBank ID: 118130322 c1; Forward 5'-CAGGATTGTTGGTCCTCAAGTG-3'; Reverse 5'-ACAGGATACAGTTACGTCAGACA-3'
Antibodyanti-SOX2 (rat monoclonal)ThermoFisherThermoFisher:14-9811-82; RRID:AB_11219471(1:1000)
Antibodyanti-BrdU (rat monoclonal)AbcamAbcam:ab6326(1:200)
Antibodyanti-TUJ1 (mouse monoclonal)BiolegendBiolegend:801202; RRID:AB_10063408(1:2000)
Antibodyanti-CTIP2 (rat monoclonal)AbcamAbcam:AB18465(1:500)
Antibodyanti-TBR2 (rabbit polyclonal)AbcamAbcam:AB23345; RRID:AB_778267(1:1000)
Antibodyanti-CC3 (rabbit polyclonal)Cell SignalingCell Signaling:9661; RRID:AB_2341188(1:250)
Antibodyanti-NEUROD2 (rabbit polyclonal)AbcamAbcam:AB104430; RRID:AB_10975628(1:500)
Antibodyanti-Ki67 (rabbit monoclonal)Cell Signaling TechnologyCell Signaling:12,202(1:1000)
Antibodyanti-PH3 (rabbit polyclonal)MilliporeMillipore:06–570(1:500)
Antibodyanti-TBR1 (rabbit monoclonal)Cell Signaling TechnologyCell Signaling Technology:49,661 S(1:1000)
Antibodyanti-DDX3X (rabbit polyclonal)Sigma AldrichSigma Aldrich:HPA001648; RRID:AB_1078635(IF, 1:500; western, 1:1000)
Antibodyanti-GFP (chicken polyclonal)AbcamAbcam:Ab13970; RRID:AB_300798(1:1000)
Antibodyanti-B-actin (mouse monoclonal)Santa CruzSanta Cruz:sc-47778(1:500)
Antibodyanti-mouse HRP (goat polyclonal)ThermoFisherThermoFisher:32430; RRID:AB_1185566(1:2000)
Antibodyanti-rabbit HRP (goat polyclonal)ThermoFisherThermoFisher:A16110; RRID:AB_2534782(1:2000)
Antibodyanti-GFP (mouse monoclonal)Santa CruzSanta Cruz:sc9996
AntibodyAlexaFluor-conjugated secondary antibodies (488, 555, 568, 594, 647)ThermoFisher(1:500)
Recombinant DNA reagentpX330-U6-Chimeric_BB-CBh-hSpCas9AddgeneAddGene:42230; RRID:Addgene_42230
Recombinant DNA reagentpX330-U6-Chimeric_BB-CBh-hSpCas9+Ddx3y guidesthis paperCloned for depletion of Ddx3y; see Figure 2
Recombinant DNA reagentpCAG-GFPPMID:32135084
Recombinant DNA reagentpCAG-GFP-human DDX3XPMID:32135084
Commercial assay or kitClick-it EdU AlexaFluor 594 imaging kitLife TechnologiesLife Technologies:c10339
Commercial assay or kitQiagen RNAeasy kitQiagenQiagen:74,034
Commercial assay or kitQiagen miRNA library prep kitQiagenQiagen:331,502
Commercial assay or kitRiboMinus Eukaryote kit v2ThermoFisherThermoFisher:A15020
Commercial assay or kitRNA Analysis Kit (15 nt)AgilentAgilent:DNF-471
Commercial assay or kitKapa mRNA HyperPrep kit with mRNA captureKapaBiosystemsKapaBiosystems:KR1352
Commercial assay or kitiScript cDNA synthesis kitBioRadBioRad:1708891
Commercial assay or kitiTaq Universal Sybr Green SupermixBioRadBioRad:1725121
Commercial assay or kitBCA protein quantificationThermoFisherThermoFisher:23,227
Commercial assay or kitECLThermoFisherThermoFisher:32,106
Chemical compound, drugEdUThermoFisherThermoFisher:A10044
Chemical compound, drugBrdUSigma AldrichSigma:B5002
Chemical compound, drugCycloheximideCalbiochemSigma:239,764
Software, algorithmFiji/ImageJPMID:22743772v1.52i
Software, algorithmQuPATHPMID:29203879v0.3.2
Software, algorithmbowtie2PMID:22388286v2.4.4
Software, algorithmSTARPMID:23104886v2.7.9a
Software, algorithmRibosomeProfilingQCDOI:10.18129/B9.bioc.ribosomeProfilingQCv1.8.0
Software, algorithmRibo-seQCDOI:10.1101/601468v0.99
Software, algorithmDESeq2PMID:25516281v1.34.0
Software, algorithmORFquantPMID:33765284v1.02
Software, algorithmRR Foundation for Statistical Computingv4.1.0
OtherVectashieldVector LabsVector Labs:H-1000–10See immunoflourescence section in methods
OtherDAPI stainThermoFisherThermoFisher:D1306See FACS section in methods
OtherPropidium iodide stainThermoFisherThermoFisher:P3566See FACS section in methods
OtherHoechst stainThermoFisherThermoFisher:H3570See immunoflourescence section in methods
OtherTurbo DNase IInvitrogenInvitrogen:AM2238See RNAseq and ribosome footprinting section in methods
OtherSuperase InInvitrogenInvitrogen:AM2694See RNAseq and ribosome footprinting section in methods
OtherTRIzolInvitrogenInvitrogen:15596026See RNAseq and ribosome footprinting section in methods
OtherGlycoBlueInvitrogenInvitrogen: AM9515See RNAseq and ribosome footprinting section in methods
OtherPNK enzymeNew England BiolabsNEB:M0247SSee RNAseq and ribosome footprinting section in methods
Other15% acrylamide denaturing urea-gelBioRadBioRad:4566053See RNAseq and ribosome footprinting section in methods
Other2 X sample dyeNovexNovex:LC6876See RNAseq and ribosome footprinting section in methods
OtherdsDNA ladderThermoFisherThermoFisher:10488023See RNAseq and ribosome footprinting section in methods
OthermiRNA laddersNew England BiolabsNEB:N2102SSee RNAseq and ribosome footprinting section in methods
OtherSYBR goldInvitrogenInvitrogen:S11494See RNAseq and ribosome footprinting section in methods
OtherSpinX columnCorningCorning:CLS8162See RNAseq and ribosome footprinting section in methods
OtherTRIzol LS reagentThermoFisherThermoFisher:10296010See RNA Immunoprecipitation section in methods
OtherLipofectamine 2000ThermoFisherThermoFisher:11668019See RNA Immunoprecipitation section in methods
OtherProtein G-coated DynabeadsThermoFisherThermoFisher:0003DSee RNA Immunoprecipitation section in methods
OtherGoTaq Green Master MixPromegaPromega:M712See RNA Immunoprecipitation section in methods
Other1 X RIPA bufferPiercePierce:89,900See SDS-PAGE and western blot analysis section in methods
Other2 X sample bufferBioRadBioRad:1610737See SDS-PAGE and western blot analysis section in methods
Otherprotease inhibitorsSigma AldrichSigma:78,429See SDS-PAGE and western blot anlaysis section in methods
Other12% polyacrylamide gelBioRadBioRad:4568046See SDS-PAGE and western blot anlaysis section in methods
OtherPVDF membraneBioRadBiorad:1704157See SDS-PAGE and western blot anlaysis section in methods

Additional files

Supplementary file 1

An excel file containing the comparison, sample size, statistical test and number of litters used (when applicable) for each data plot.

https://cdn.elifesciences.org/articles/78203/elife-78203-supp1-v1.xlsx
Supplementary file 2

An excel file containing the primer and sgRNA sequences used in this paper.

https://cdn.elifesciences.org/articles/78203/elife-78203-supp2-v1.xlsx
Supplementary file 3

An excel file containing three tabs (1) the differential expression analysis of controls versus Ddx3x cKO for RNAseq and Riboseq data (using only unique reads); (2) the analysis of translational efficiency from wildtype (ie: control) data (using all reads); (3) the cell type-enriched transcripts used in Figure 6B.

https://cdn.elifesciences.org/articles/78203/elife-78203-supp3-v1.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/78203/elife-78203-transrepform1-v1.docx

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