Abstract

Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts1, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation2. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage2: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells. This enables analysis, in mature lymphocytes, of the functions of genes that are required more generally for mouse development3. Blastocyst complementation has been extended to pancreas organogenesis4, and used to generate several other tissues or organs5,6,7,8,9,10, but an equivalent approach for brain organogenesis has not yet been achieved. Here we describe neural blastocyst complementation (NBC), which can be used to study the development and function of specific forebrain regions. NBC involves targeted ablation, mediated by diphtheria toxin subunit A, of host-derived dorsal telencephalic progenitors during development. This ablation creates a vacant forebrain niche in host embryos that results in agenesis of the cerebral cortex and hippocampus. Injection of donor ES cells into blastocysts with forebrain-specific targeting of diphtheria toxin subunit A enables donor-derived dorsal telencephalic progenitors to populate the vacant niche in the host embryos, giving rise to neocortices and hippocampi that are morphologically and neurologically normal with respect to learning and memory formation. Moreover, doublecortin-deficient ES cells—generated via a CRISPR–Cas9 approach—produced NBC chimaeras that faithfully recapitulated the phenotype of conventional, germline doublecortin-deficient mice. We conclude that NBC is a rapid and efficient approach to generate complex mouse models for studying forebrain functions; this approach could more broadly facilitate organogenesis based on blastocyst complementation.

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All data generated or analysed during this study are included in this manuscript and its Supplementary Information.

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Acknowledgements

We thank members of the Alt laboratory and C. A. Walsh for stimulating discussions, P.-Y. Huang for help with blastocyst injections, H.-L. Cheng for advice and help with ES cell culture, and S. V. Griswold and T. Chari for assistance with behavioural experiments. Behavioural testing was carried out at the Boston Children’s Hospital (BCH) Neurodevelopmental Behavior Core (CHB IDDRC, 1U54HD090255). This work was supported by the Howard Hughes Medical Institute, the BCH Department of Medicine (DOM) Support Fund, and the BCH DOM Anderson Porter Fund and a major grant from the Charles H. Hood Foundation. B.S. is a Kimmel Scholar of The Sidney Kimmel Foundation, supported by NIA/NIH grant AG043630, the UCSF Brain Tumor SPORE Career Development Program, the American Cancer Society, the Andrew McDonough B+ Foundation, the Shurl and Kay Curci Foundation and a Martin D. Abeloff V Scholar award of The V Foundation for Cancer Research. B.S. also holds the Suzanne Marie Haderle and Robert Vincent Haderle Endowed Chair, UCSF. H.-Q.D. is a fellow of the Cancer Research Institute of New York. F.W.A. is an investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. These authors contributed equally: Amelia N. Chang, Zhuoyi Liang, Hai-Qiang Dai

  2. These authors jointly supervised this work: Bjoern Schwer, Frederick W. Alt

Affiliations

  1. Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics and Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    • Amelia N. Chang
    • , Zhuoyi Liang
    • , Hai-Qiang Dai
    • , Aimee M. Chapdelaine-Williams
    •  & Frederick W. Alt
  2. Division of Neurology, Kirby Center for Neurobiology, Boston Children’s Hospital, Boston, MA, USA

    • Nick Andrews
  3. Rodent Histopathology Core, Harvard Medical School, Boston, MA, USA

    • Roderick T. Bronson
  4. Department of Neurological Surgery and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA, USA

    • Bjoern Schwer

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Contributions

A.N.C., B.S. and F.W.A. designed the study; A.N.C., H.-Q.D., Z.L. and B.S. performed experiments; A.M.C.-W. performed NBC injections and related mouse work; B.S. and F.W.A. supervised the research. A.N.C., H-Q.D., Z.L., B.S. and F.W.A. wrote the paper. R.T.B. reviewed histology sections; N.A. provided advice and carried out behavioural experiments.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Bjoern Schwer or Frederick W. Alt.

Extended data figures and tables

  1. Extended Data Fig. 1 NBC chimaeras have normal overall appearance, postnatal growth, body weights and survival rates.

    a, Representative photograph of newborn mice. From left to right: wild-type non-chimeric pup from a normal mouse breeding cross; conventional chimaera generated by injecting donor ES cells into a wild-type host blastocyst; NBC chimaera without reconstitution of the forebrain using donor ES cells (‘ablated’); and NBC chimaera with reconstitution of the forebrain using donor ES cells (‘reconstituted’). Note the abnormal head curvature of the ablated NBC chimaera relative to the other mice (black asterisks), consistent with the lack of a forebrain. Also note the lack of a milk spot (blue asterisks), consistent with the inability to suckle as a result of incomplete jaw development. At least three mice per group from independent experiments were analysed with similar results. b, Body-weight gain of conventional and NBC chimaeras with normal jaws (n = 7 each) in the first postnatal week. Data represent mean ± s.d.; not significant, P > 0.05 (multiple unpaired, two-tailed t-tests, Holm–Sidak post hoc correction) for all data points. c, Body weights of 2–3-month-old male conventional (n = 18) and NBC (n = 16) chimaeras. Data represent mean and s.d.; NS, not significant, P > 0.05 (unpaired, two-tailed t test). d, Postnatal survival rate of conventional (n = 16 males) and NBC (n = 16 males) chimaeras; NS, not significant; P > 0.05 (two-sided log-Rank, Mantel–Cox test. #One conventional chimaera was euthanized because of a genital mass. ##Four NBC chimaeras died because their cage flooded; all other mice were euthanized at 24 months of age.

  2. Extended Data Fig. 2 Generation of Emx1-cre;R26-DTA;DsRed.T3 NBC host blastocysts and eGFP-labelled donor ES cells.

    a, Schematic of the breeding crosses used to generate DsRed.T3-labelled NBC host blastocysts. Tg, transgene. b, Representative bright field and fluorescence microscopy images of wild-type TC1 ES cells before and after lentiviral integration of H2B–eGFP. The ES cells shown stably express H2B–eGFP and were repeatedly imaged during the study with similar results.

  3. Extended Data Fig. 3 NBC chimaeras show variable donor contribution in non-brain tissues, as do conventional chimaeras.

    a, Representative images showing the extent of H2B–eGFP-expressing wild-type (TC1) donor ES cell (ESC) contribution to the indicated non-brain tissues in conventional (conv.) and NBC chimaeras at P0, depicting donor-derived cells in green and host-derived cells in red. Nuclei were visualized by DAPI (blue). These experiments were performed on six mice (n = 3, NBC chimaeras; n = 3, conventional chimaeras). b, Quantification of donor ES cell contribution in indicated tissues of conventional and NBC chimaeras (n = 3 mice each). Data represent mean and s.d. The variance in donor contribution was not significantly different between conventional and NBC chimaeras for all tissues examined, consistent with competition between donor and host cells in these non-brain tissues in both types of chimaeras (NS, not significant, P > 0.05; F test for equality of variances). Scale bar, 10 μm.

  4. Extended Data Fig. 4 Brains of NBC mice are structurally normal.

    a, b, Cell numbers in the indicated somatosensory cortical layers (a) and hippocampal regions (b) in P7 conventional (black) and NBC (orange) chimaeras (n = 6 mice each) generated by blastocyst injection of EF1 ES cells. Cortical layers II–IV and V were identified by Cux1 and Ctip2 immunoreactivity, respectively. Nuclei were visualized with DAPI. Hip, hippocampus. c, d, Width of the indicated somatosensory cortical layers (c) and hippocampal regions (d) in P7 conventional (black) and NBC (blue) chimaeras generated by blastocyst injection of TC1-derived ES cells (n = 4 mice each), analysed as in Fig. 3. e, f, Cell numbers in the indicated somatosensory cortical layers (e) and hippocampal regions (f) in P7 conventional (black) and NBC (blue) chimaeras generated by blastocyst injection of TC1-derived ES cells (n = 4 mice each), analysed as in a, b. g, Dentate gyrus measurements in P0 conventional chimaeras (n = 3; black) and NBC chimaeras (n = 4; blue) generated by blastocyst injection of H2B–eGFP-expressing TC1 ES cells. For panels a–g, horizontal bars indicate mean; error bars denote s.e.m.; NS, not significant, P > 0.05 (unpaired, two-tailed t test). h, i, Representative images of coronal somatosensory cortex sections at P7 stained for Satb2 (h, red), Foxp2 (i, red) and DAPI (blue). These experiments were repeated on six mice (n = 3, NBC chimaeras; n = 3, conventional chimaeras). Scale bars, 50 μm.

  5. Extended Data Fig. 5 NBC mice have normal proportions of non-neuronal cells and do not show signs of neuroinflammation.

    a, Representative immunofluorescence images of oligodendrocytes (Olig2), astrocytes (GFAP) and microglia (Iba1) in cortical brain sections of conventional and NBC chimaeras, with a wild-type non-chimeric mouse for comparison. The non-neuronal cells are shown in red; DAPI-stained nuclei are in blue. These experiments were repeated on 9 mice (n = 3, NBC chimaeras; n = 3, conventional chimaeras; n = 3, non-chimeric wild-type mice). b, Quantification of Olig2-, GFAP- or Iba1-positive cells in cortical brain sections of conventional chimaeras, NBC chimaeras and wild-type non-chimeric mice (n = 3 each). Data represent mean and s.e.m.; NS, not significant, P > 0.05 (one-way ANOVA, Tukey’s post hoc correction for multiple comparisons). Scale bar, 10 μm.

  6. Extended Data Fig. 6 Brains of NBC mice are functionally normal.

    a, Schematic of the novel-object recognition task. b, Locomotor activity of male two-month-old conventional (n = 15) or NBC (n = 14) chimaeras during the habituation phase of the novel-object recognition paradigm. Data represent mean and s.e.m.; NS, P > 0.05 (unpaired two-tailed t-test). c, Latency to platform for conventional (black) or NBC (orange) chimaeras in the indicated visual and learning trials of the Morris water maze task. Male two-month-old conventional and NBC chimaeras (n = 16 mice each) were analysed. Data represent mean and s.e.m.; no significant difference was observed between conventional and NBC chimaeras across the visual trials (F(1, 30) = 0.223, P = 0.640) or learning trials (F(1, 30) = 0.296, P = 0.590) (two-way mixed-model ANOVA). A significant difference was observed in latency over the learning trials, indicating that both groups of mice are able to learn (F(1.627, 48.815) = 29.426, P < 0.0005) (two-way mixed-model ANOVA with Greenhouse–Geisser correction for violation of sphericity). d, Path length traversed in each quadrant by conventional and NBC chimaeras during the probe trial. Male two-month-old conventional and NBC chimaeras (n = 16 mice each) were analysed. Data represent mean and s.e.m.; no significant difference between conventional and NBC chimaeras (F(1, 30) = 1.433, P = 0.241) (two-way mixed-model ANOVA). Significant preference was observed for the north-east (NE) quadrant where the platform had previously been located, indicating memory retention of the prior platform location; ***P < 0.001 (multiple pairwise comparisons with Bonferroni post hoc correction).

  7. Extended Data Fig. 7 Generation of Dcx−/Y NBC chimaeras.

    a, Top, schematic of Dcx inactivation. sgRNA target sequences (red and orange arrowheads) flanking exon 2 (ex 2) and exon 3 (ex 3) are indicated. Probes (black rectangles) and restriction enzyme sites (P, PstI; B, BglII) used for Southern blotting are indicated. Predicted restriction fragment sizes are indicated in kb. Bottom, predicted Dcx exon 2 and exon 3 deletion (Δ) locus. b, Southern blot analysis of genomic DNA from parental (P) Dcx+/Y (wild-type) ES cell line and Dcx−/Y ES-cell clones 7F and 8E, using the probes and digests depicted in a. Southern blot analysis was performed five times. c, Sequence analysis of Dcx deletion junctions (black arrowheads); sgRNA target sequences flanking the deletion junction are denoted in red and orange. Both of the Dcx−/Y ES cell clones have a unique junction sequence, which suggests that each derived from an independent deletion event. d, Western blotting of whole-cell lysates from cortex or hippocampus of wild-type or Dcx−/Y NBC chimaeras at P0 with antibodies to Dcx (rabbit anti-Dcx) and βIII-tubulin as a loading control. Each lane (1–9) depicts cortex or hippocampus whole-cell lysate from one mouse; experiments were performed on nine mice (n = 4, Dcx−/Y ES-cell clone 8E; n = 5, wild-type ES cells) e, Representative immunofluorescence images of Dcx expression in cortex and hippocampus of wild-type or Dcx−/Y chimaeras at P0. Twenty-one mice (n = 6, Dcx−/Y ES-cell clone 7F; n = 4 Dcx−/Y ES-cell clone 8E; n = 11 wild-type ES cells) were analysed. Dcx-expressing cells (red) were detected with rabbit or goat Dcx antibodies. Nuclei were DAPI-stained (blue). f, Representative images of somatosensory cortical brain sections (acquired between layers IV and V) from P0 Dcx−/Y chimaeras co-stained with Dcx (red) and GABA (green) antibodies. Merged images are shown on the right. Nuclei were DAPI-stained (blue). Solid white arrowheads indicate cells that are double-positive for Dcx and GABA (host-derived immature interneurons). Hollow arrowheads indicate GABA single-positive cells, which are either donor-derived mature interneurons, donor-derived immature interneurons or host-derived mature interneurons. g, Representative images of somatosensory cortical brain sections (acquired between layers IV and V) from P0 Dcx−/Y chimaeras co-stained with Dcx (red) and GAD67 (green) antibodies. Merged images are shown on the right. Nuclei were DAPI-stained (blue). Solid white arrowheads indicate host-derived immature interneurons, whereas hollow arrowheads indicate cells that are either donor-derived mature interneurons, donor-derived immature interneurons or host-derived mature interneurons. For experiments in f, g, nine mice (n = 4, Dcx−/Y ES-cell clone 8E; n = 5 wild-type ES cells) were analysed. h, Representative images of somatosensory cortical brain sections from P0 wild-type and Dcx−/Y chimaeras stained with GABA antibodies (green). Nuclei were DAPI-stained (blue). Seven mice were analysed (n = 3, NBC chimaeras; n = 4, conventional chimaeras). i, Cell density of GABA-positive interneurons in somatosensory cortex from control chimaeras (n = 4 mice) and Dcx−/Y chimaeras (n = 3 mice). Centre denotes mean, error bars indicate s.e.m. NS, not significant, P > 0.05 (unpaired, two-tailed t-test). j, Mean ( ± s.d.) body weights of wild-type (TC1) or Dcx−/Y chimaeras; NS, not significant, P > 0.05 (one-way ANOVA, Tukey’s post hoc correction). For P0 cohort, n = 6 for Dcx 7F; n = 5 for Dcx 8E; n = 11 for wild-type TC1 controls. For two-month-old cohort, n = 6 for Dcx 7F; n = 8 for Dcx 8E; n = 5 for wild-type TC1 controls. Scale bars, 10 μm (f, g), 50 μm (h).

  8. Extended Data Fig. 8 Dcx−/Y NBC chimaeras show a mutant hippocampal phenotype.

    a, Representative images of H&E-stained coronal sections of hippocampus from two-month-old wild-type chimaeras and Dcx−/Y chimaeras. Dashed lines highlight the CA3 region, where the stratum pyramidale is present as a single layer in wild-type chimaeras but is abnormally divided into an internal and external layer in the Dcx−/Y chimaeras. Figure 4a provides a schematic of the hippocampus. b, Enlarged, representative images of the CA3 region in two two-month-old wild-type and Dcx−/Y chimaeras. For experiments in a, b, 14 2-month-old Dcx−/Y chimaeras—generated from 2 different Dcx−/Y ES clones (n = 6 from clone Dcx 7F; n = 8 from clone Dcx 8E)—and 5 wild-type chimaeras were analysed. All 14 Dcx−/Y chimaeras showed the hippocampal mutant phenotype. c, Representative bright field and fluorescence microscopy images of Dcx−/Y ES cells before and after lentiviral integration of H2B–eGFP. d, Representative images from experiments performed on six mice (n = 3, NBC chimaeras; n = 3, conventional chimaeras) showing the extent of the contribution of Dcx−/Y H2B–eGFP-expressing donor ES cells to indicated brain regions in conventional and NBC chimaeras at P0 (green, cells derived from donor ES cells; red, host-derived cells; blue, DAPI-stained nuclei). e, Quantification of the contribution of Dcx−/Y H2B–eGFP-expressing donor ES cells in conventional and NBC chimaeras (n = 3 mice each). Data are mean ± s.d. Variance was significantly different between conventional and NBC chimaeras for CA1, CA3 and cortex (F test for equality of variances), which reflects the wide variation in donor contribution among individual conventional chimaeras in contrast to consistently high donor contribution in NBC chimaeras. No difference was observed for midbrain (NS, not significant; P = 0.7812), which is consistent with competition between donor and host cells in this non-ablated brain region in both types of chimaera. f, Quantification of variance in contribution of Dcx−/Y H2B–eGFP-expressing donor ES cells in non-brain tissues, relative to hippocampus in NBC chimaeras (n = 4). Donor contribution in the hippocampus was calculated as the average of CA1 and CA3 values. Data are mean ± s.d. (F test for equality of variances). g. Quantification of Dcx mutant hippocampal phenotype in conventional chimaeras (n = 12) and NBC chimaeras (n = 13) at P0. Dcx phenotype scores were determined by reviewing a series of coronal brain step-sections from each chimaera and determining the number of sections that show a clear CA3 double layer (score = 1), some degree of CA3 disorganization (score = 0.5) or normal CA3 layer morphology (score = 0). Mean Dcx phenotype scores across all sections of a given chimaera are plotted. Each data point corresponds to one chimaera. Horizontal lines indicate mean phenotype score ( ± s.d.) across all chimaeras per group. Conventional chimaeras show significant variation in the severity of the mutant phenotype, in contrast to the NBC chimaeras (F test for equality of variances). h, Widths of somatosensory cortex layers in P0 conventional (n = 3, black) or NBC (n = 3, orange) chimaeras injected with Dcx−/Y H2B–eGFP-expressing ES cells, determined by DAPI and Ctip2 staining. Centre denotes mean, error bars indicate s.e.m. NS, not significant, P > 0.05 (two-way ANOVA with Bonferroni post hoc correction for multiple comparisons). i, Dentate gyrus measurements in conventional chimaeras (n = 3; black) and NBC chimaeras (n = 4; orange) at P0, generated by blastocyst injection of Dcx−/Y H2B–eGFP-expressing ES cells. Horizontal bars indicate mean; error bars denote s.e.m.; NS, not significant, P > 0.05 (unpaired, two-tailed t-test). Scale bars, 100 μm (a, b), 10 μm (d).

  9. Extended Data Fig. 9 Rates of recovery, survival, and litter sizes across multiple pregnancies and ES cell lines.

    a, Average recovery rate ( ± s.e.m.) of liveborn pups from wild-type blastocysts without ES cell injection or injected with wild-type ES cells (EF1 and TC1). NS, not significant, P > 0.05 (one-way ANOVA with Tukey’s post hoc correction for multiple comparisons). Number of transfers (foster pregnancies) and pup survival rate at P0 is listed above the graph. b, Mean litter size ( ± s.e.m.) per foster recipient of wild-type blastocysts without ES cell injection or injected with wild-type ES cells (EF1 and TC1); NS, not significant, P > 0.05 (one-way ANOVA, Tukey’s post hoc correction for multiple comparisons). c, Average recovery rate ( ± s.e.m.) of liveborn pups from NBC blastocysts without ES cell injection or injected with wild-type (EF1 and TC1) or Dcx−/Y ES cells. NS, not significant, P > 0.05 (one-way ANOVA with Tukey’s post hoc correction for multiple comparisons). Number of transfers (foster pregnancies) and pup survival rate at P0 is listed above the graph. d, Mean litter size ( ± s.e.m.) per foster recipient of NBC blastocysts without ES cell injection or injected with wild-type (EF1 and TC1) or Dcx−/Y ES cells; NS, not significant, P > 0.05 (one-way ANOVA, Tukey’s post hoc correction for multiple comparisons). e, Average recovery rate ( ± s.e.m.) of mice from mixed wild-type and NBC blastocyst transfers; NS, not significant, P > 0.05 (unpaired, two-tailed t-test). f, Mean litter size ( ± s.e.m.) per foster recipient; NS, not significant, P > 0.05 (unpaired, two-tailed t-test). For experiments in e, f, mice were inter-crossed to simultaneously generate wild-type or NBC blastocysts with or without expression of DsRed.T3 (see schematic in Extended Data Fig. 2a) and were injected with either wild-type (TC1) H2B–eGFP-expressing or Dcx−/Y H2B–eGFP-expressing ES cells, followed by transfer into fosters. Pups were then genotyped to determine whether they were wild-type or derived from NBC blastocysts.

Supplementary information

  1. Reporting Summary

  2. Supplementary Table 1

    NBC chimaeras show normal SHIRPA phenotypic assessment scores. Basic autonomic, neurological, and sensory functions of NBC chimaeras (n=16) and conventional chimaera controls (n=18) were scored using a modified SHIRPA phenotypic assessment. Male mice between 2-3 months of age were tested. No statistically significant differences in mean SHIRPA scores between groups were detected; P>0.05 (unpaired, two-tailed t test).

  3. Supplementary Table 2

    Sequences and description of oligonucleotides used in this study.

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https://doi.org/10.1038/s41586-018-0586-0

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