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. 2022 Jul 29;8(30):eabn7702.
doi: 10.1126/sciadv.abn7702. Epub 2022 Jul 29.

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development

Felipe Mora-Bermúdez  1   2 , Philipp Kanis  2 , Dominik Macak  2 , Jula Peters  1 , Ronald Naumann  1 , Lei Xing  1 , Mihail Sarov  1 , Sylke Winkler  1 , Christina Eugster Oegema  1 , Christiane Haffner  1 , Pauline Wimberger  3 , Stephan Riesenberg  2 , Tomislav Maricic  2 , Wieland B Huttner  1 , Svante Pääbo  2   4
Affiliations

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development

Felipe Mora-Bermúdez et al. Sci Adv. .

Abstract

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

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Figures

Fig. 1.
Fig. 1.. More SAC-positive kinetochores in modern human than chimpanzee APs.
(A to C) APs in metaphase stained with DAPI (4′,6-diamidino-2-phenylindole) and immunostained for the SAC marker BubR1 (red in merges) and the kinetochore marker CREST (green in merges). Arrowheads indicate overlap of BubR1 and CREST immunoreactivity within the metaphase plate. (A) GW12 human neocortex; (B) day 30 human iPSC-derived cerebral organoid; and (C) day 30 chimpanzee iPSC-derived cerebral organoid. White dashed lines, ventricular surface. Scale bar, 5 μm. (D) Quantification of kinetochores positive for BubR1 per AP metaphase plate, for the tissues in (A) to (C) (GW11 to GW12, days 30 to 32). Data are the means ± SEM of ≥41 APs from ≥3 independent experiments each, with 3 neocortex samples and ≥5 organoids each. Brackets with **P < 0.01; ***P < 0.001; n. s., nonsignificant (Kruskal-Wallis test with Dunn’s multiple comparisons correction).
Fig. 2.
Fig. 2.. Fewer lagging chromosomes in modern human than chimpanzee APs.
(A to D) Mitotic APs in days 30 to 32 modern human iPSC-derived cerebral organoids stained with DAPI. Examples of APs in anaphase (A) and telophase (B) without lagging chromosomes, and in anaphase (C) and telophase (D) with lagging chromosomes, or large chromosome fragments (arrowheads). White dashed lines, ventricular surface. Scale bar, 5 μm. (E) Cumulative quantification (see Materials and Methods) of the percentages of AP divisions with lagging chromosomes in days 30 to 32 modern human (grayscale) and chimpanzee (Chimp, green) iPSC-derived cerebral organoids. Data are the sum from ≥5 independent experiments and a total of ≥342 AP divisions (only anaphase or telophase) each. Each bar shows the data for the sum of mitotic APs in anaphase plus telophase, with the stacked percentages for telophase (dark shade) and anaphase (light shade) being indicated separately.
Fig. 3.
Fig. 3.. Mice humanized for KIF18a, KNL1, and SPAG5 reveal genes involved in metaphase prolongation.
(A) Schematic representation of the domain structure of KNL1, KIF18a, and SPAG5, carrying a total of six amino acid (aa) residue differences between modern humans (h) and archaic humans (a): KNL1, Ha159Rh, and Ga1086Sh; KIF18a, Ra67Kh; SPAG5, Pa43Sh, Ea162Gh, and Da410Hh. Note that in wt mice, these six amino acid residues are identical to those in archaic humans and apes. (B) C57Bl6/NCrl wt mice were gene edited using CRISPR-Cas9 (see Materials and Methods) to change the wt (Neanderthal-like) amino acid residue(s) specified in (A) to the modern human variants. Five different humanized (h) mouse lines were generated as indicated. (C to H) Live-tissue imaging of the indicated mitotic phases of APs in organotypic coronal slice cultures of neocortex of E11.5 mice of the indicated genotypes (see Materials and Methods). Zero (0) minute is metaphase plate onset. Time-lapse intervals are 1.5 min. Red lines indicate the duration of metaphase, white dashed lines the ventricular surface. Scale bar, 5 μm. (I) Times between the start of chromosome congression and metaphase plate onset (referred to as “Congression” or “Prometaphase”); (J) between metaphase plate onset and chromatid segregation onset (referred to as “Metaphase plate” or “Metaphase”); and (K) between the onset of chromatid segregation and onset of chromosome decondensation (referred to as “Anaphase”), for APs in the six neocortical tissues described in (C) to (H). Data are the means ± SEM of ≥50 APs from ≥3 independent experiments, with a total of ≥5 neocortices, for each of the six lines. Brackets with *P < 0.05; ***P < 0.001 (Kruskal-Wallis test with Dunn’s multiple comparisons correction).
Fig. 4.
Fig. 4.. APs in human organoids ancestralized for KIF18a and KNL1 have a shorter metaphase than modern human controls.
(A) Human H9 ESCs were gene edited using CRISPR technology to convert the indicated three amino acids in KIF18a and KNL1 from the modern human (h) to the ancestral (a) Neanderthal-like states, and used to grow cerebral organoids (see Materials and Methods). (B and C) Live-tissue imaging of the indicated mitotic phases of APs in organotypic slice cultures of days 27 to 30 cerebral organoids grown from the indicated control (B) and ancestralized (C) line. Zero (0) minute is metaphase plate onset. Time-lapse intervals are 2.08 min. Red lines indicate the duration of metaphase, white dashed lines the ventricular surface. Scale bar, 5 μm. (D) Times between the start of chromosome congression and metaphase plate onset (referred to as Congression), between metaphase plate onset and chromatid segregation onset (referred to as “Metaphase plate”), and between chromatid segregation onset and general chromosome decondensation onset (referred to as “Anaphase”), for APs in the two types of organoids as described for line 1 in (B) and (C). Data are the means ± SEM for organoids grown from the two control and the two ancestralized lines (L1, L2) and comprise ≥119 APs from four independent experiments, with ≥7 organoids, for each of the lines. Bracket with ****P < 0.0001 (Mann-Whitney U test).
Fig. 5.
Fig. 5.. More SAC-positive kinetochores in humanized mouse APs and fewer SAC-positive kinetochores in ancestralized human organoid APs.
(A, B, D, and E) APs in metaphase stained with DAPI and immunostained for BubR1 (red in merges) and the kinetochore marker CREST (green in merges). Arrowheads indicate overlap of BubR1 and CREST immunoreactivity within the metaphase plate. (A) Neocortex of E11.5 wt (ancestral-like) mouse; (B) neocortex of E11.5 mouse humanized for Kif18a and Knl1; (D) modern human nonedited day 27 cerebral organoid (control line 1); (E) day 29 organoid ancestralized for KIF18a and KNL1 (edited line 1). White dashed lines, ventricular surface. Scale bar, 5 μm. (C) Number of BubR1-positive kinetochores per AP metaphase plate in mouse neocortex as described in (A) and (B). Data are the means ± SEM of ≥41 APs from three independent experiments, with a total of ≥5 neocortices, for each of the two types of mice. Bracket with **P < 0.01 (Mann-Whitney U test). (F) Number of BubR1-positive kinetochores per AP metaphase plate for days 27 to 30 organoids (e.g., D and E). Data are the means ± SEM of ≥54 APs from six independent experiments, with a total of ≥10 organoids, for each of the two ESC lines. Bracket with ***P < 0.001 (Mann-Whitney U test).
Fig. 6.
Fig. 6.. Fewer lagging chromosomes in humanized mouse APs, and more lagging chromosomes in ancestralized human organoid APs.
(A to H) Mitotic APs stained with DAPI in the neocortex of E11.5 wt mice and mice carrying humanized hKif18a-hKnl1 (A to D) and in days 27 to 30 control cerebral organoids (line 2) and organoids with ancestralized aKIF18a-aKNL1 (line 2, E to H). Examples of APs in anaphase (A and E) and telophase (B and F) without lagging chromosomes, and in anaphase (C and G) and telophase (D and H) with lagging chromosomes, or large chromosome fragments (arrowheads). White dashed lines, ventricular surface. Scale bar, 5 μm. (I) Cumulative quantification (see Materials and Methods) of the percentages of AP divisions with lagging chromosomes for the tissues described in (A) to (H), as well as for the neocortex of E11.5 mice carrying humanized hKif18a-hKnl1-hSpag5. The mouse data are the sum of ≥314 AP divisions in anaphase or telophase from six experiments for each of the three types of mouse neocortex, and the human organoid data are the sum of ≥389 AP divisions from six experiments for each of the two types of organoids (sums of lines 1 and 2). The percentages for telophase (dark shade) and anaphase (light shade) are indicated separately.
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