Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.

Brain Research Institute University of Zurich Zurich Switzerland

Department of Biosystems Science and Engineering ETH Zürich Basel Switzerland

Department of Chemistry Biochemistry and Pharmaceutical Sciences University of Bern Bern Switzerland

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Brno Czech Republic

Department of Molecular Life Sciences University of Zurich Zurich Switzerland

Department of Neurodegenerative Disease UCL Institute of Neurology London UK

Department of Quantitative Biomedicine University of Zurich Zurich Switzerland

Institute of Neuropathology University of Zurich Zurich Switzerland

MaxWell Biosystems AG Zurich Switzerland

NCCR RNA and Disease Technology Platform Bern Switzerland

Queen Square Brain Bank for Neurological diseases Department of Movement Disorders UCL Institute of Neurology London UK

SIB Swiss Institute of Bioinformatics University of Zurich Zurich Switzerland

Zobrazit více v PubMed

Neumann M, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006;314:130–133. doi: 10.1126/science.1134108. PubMed DOI

Brown A-L, et al. TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature. 2022;603:131–137. doi: 10.1038/s41586-022-04436-3. PubMed DOI PMC

Klim JR, et al. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat. Neurosci. 2019;22:167–179. doi: 10.1038/s41593-018-0300-4. PubMed DOI PMC

Ma XR, et al. TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A. Nature. 2022;603:124–130. doi: 10.1038/s41586-022-04424-7. PubMed DOI PMC

Melamed Z, et al. Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat. Neurosci. 2019;22:180–190. doi: 10.1038/s41593-018-0293-z. PubMed DOI PMC

Bohaciakova D, et al. A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors. Stem Cell Res. Ther. 2019;10:83. doi: 10.1186/s13287-019-1163-7. PubMed DOI PMC

Lam M, et al. Single-cell study of neural stem cells derived from human iPSCs reveals distinct progenitor populations with neurogenic and gliogenic potential. Genes Cells. 2019;24:836–847. doi: 10.1111/gtc.12731. PubMed DOI PMC

Kanton S, et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature. 2019;574:418–422. doi: 10.1038/s41586-019-1654-9. PubMed DOI

Arai T, et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 2006;351:602–611. doi: 10.1016/j.bbrc.2006.10.093. PubMed DOI

Sephton CF, et al. TDP-43 is a developmentally regulated protein essential for early embryonic development. J. Biol. Chem. 2010;285:6826–6834. doi: 10.1074/jbc.M109.061846. PubMed DOI PMC

Polymenidou M, et al. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat. Neurosci. 2011;14:459–468. doi: 10.1038/nn.2779. PubMed DOI PMC

Ayala YM, et al. TDP-43 regulates its mRNA levels through a negative feedback loop. EMBO J. 2011;30:277–288. doi: 10.1038/emboj.2010.310. PubMed DOI PMC

Tollervey JR, et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat. Neurosci. 2011;14:452–458. doi: 10.1038/nn.2778. PubMed DOI PMC

Laferrière F, et al. TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates. Nat. Neurosci. 2019;22:65–77. doi: 10.1038/s41593-018-0294-y. PubMed DOI

Porta S, et al. Distinct brain-derived TDP-43 strains from FTLD-TDP subtypes induce diverse morphological TDP-43 aggregates and spreading patterns in vitro and in vivo. Neuropathol. Appl. Neurobiol. 2021;47:1033–1049. doi: 10.1111/nan.12732. PubMed DOI PMC

De Rossi P, et al. FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade. EMBO Rep. 2021;22:e53877. doi: 10.15252/embr.202153877. PubMed DOI PMC

Lagier-Tourenne C, Polymenidou M, Cleveland DW. TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum. Mol. Genet. 2010;19:R46–64. doi: 10.1093/hmg/ddq137. PubMed DOI PMC

Ling S-C, Polymenidou M, Cleveland DW. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron. 2013;79:416–438. doi: 10.1016/j.neuron.2013.07.033. PubMed DOI PMC

Arnold ES, et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43. Proc. Natl Acad. Sci. USA. 2013;110:E736–E745. doi: 10.1073/pnas.1222809110. PubMed DOI PMC

Ling JP, Pletnikova O, Troncoso JC, Wong PC. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015;349:650–655. doi: 10.1126/science.aab0983. PubMed DOI PMC

Kiskinis E, et al. Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1. Cell Stem Cell. 2014;14:781–795. doi: 10.1016/j.stem.2014.03.004. PubMed DOI PMC

Wainger BJ, et al. Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Rep. 2014;7:1–11. doi: 10.1016/j.celrep.2014.03.019. PubMed DOI PMC

Ho R, et al. Cross-comparison of human iPSC motor neuron models of familial and sporadic ALS reveals early and convergent transcriptomic disease signatures. Cell Syst. 2021;12:159–175.e9. doi: 10.1016/j.cels.2020.10.010. PubMed DOI PMC

Workman MJ, et al. Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects. Neuron. 2023;111:1191–1204.e5. doi: 10.1016/j.neuron.2023.01.010. PubMed DOI PMC

Ratti A, et al. Chronic stress induces formation of stress granules and pathological TDP-43 aggregates in human ALS fibroblasts and iPSC-motoneurons. Neurobiol. Dis. 2020;145:105051. doi: 10.1016/j.nbd.2020.105051. PubMed DOI

Lee S, Huang EJ. Modeling ALS and FTD with iPSC-derived neurons. Brain Res. 2017;1656:88–97. doi: 10.1016/j.brainres.2015.10.003. PubMed DOI PMC

Hock E-M, et al. Hypertonic stress causes cytoplasmic translocation of neuronal, but not astrocytic, FUS due to impaired transportin function. Cell Rep. 2018;24:987–1000.e7. doi: 10.1016/j.celrep.2018.06.094. PubMed DOI

Emmenegger M, et al. LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies. EMBO Mol. Med. 2021;13:e14745. doi: 10.15252/emmm.202114745. PubMed DOI PMC

Sahadevan S, et al. Synaptic FUS accumulation triggers early misregulation of synaptic RNAs in a mouse model of ALS. Nat. Commun. 2021;12:3027. doi: 10.1038/s41467-021-23188-8. PubMed DOI PMC

Ballini M, et al. A 1,024-channel CMOS microelectrode array with 26,400 electrodes for recording and stimulation of electrogenic cells in vitro. IEEE J. Solid State Circuits. 2014;49:2705–2719. doi: 10.1109/JSSC.2014.2359219. PubMed DOI PMC

Müller J, et al. High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels. Lab Chip. 2015;15:2767–2780. doi: 10.1039/C5LC00133A. PubMed DOI PMC

Ronchi S, et al. Microelectrode arrays: electrophysiological phenotype characterization of human iPSC‐derived neuronal cell lines by means of high‐density microelectrode arrays. Adv. Biol. 2021;5:2170031. doi: 10.1002/adbi.202170031. PubMed DOI PMC

Buccino AP, et al. SpikeInterface, a unified framework for spike sorting. eLife. 2020;9:e61834. doi: 10.7554/eLife.61834. PubMed DOI PMC

Polioudakis D, et al. A single-cell transcriptomic atlas of human neocortical development during mid-gestation. Neuron. 2019;103:785–801.e8. doi: 10.1016/j.neuron.2019.06.011. PubMed DOI PMC

Franzén O, Gan L-M, Björkegren JLM. PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database. 2019;2019:baz046. doi: 10.1093/database/baz046. PubMed DOI PMC

Liu EY, Russ J, Lee EB. Neuronal transcriptome from C9orf72 repeat expanded human tissue is associated with loss of C9orf72 function. Free Neuropathol. 2020;1:23. PubMed PMC

Markusic D, Oude-Elferink R, Das AT, Berkhout B, Seppen J. Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter. Nucleic Acids Res. 2005;33:e63. doi: 10.1093/nar/gni062. PubMed DOI PMC

Liu EY, et al. Loss of nuclear TDP-43 is associated with decondensation of LINE retrotransposons. Cell Rep. 2019;27:1409–1421.e6. doi: 10.1016/j.celrep.2019.04.003. PubMed DOI PMC

Lukavsky PJ, et al. Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43. Nat. Struct. Mol. Biol. 2013;20:1443–1449. doi: 10.1038/nsmb.2698. PubMed DOI

Avendaño-Vázquez SE, et al. Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection. Genes Dev. 2012;26:1679–1684. doi: 10.1101/gad.194829.112. PubMed DOI PMC

Pérez-Berlanga M, et al. Loss of TDP-43 oligomerization or RNA binding elicits distinct aggregation patterns. EMBO J. 2023;42:e111719. doi: 10.15252/embj.2022111719. PubMed DOI PMC

Fratta P, et al. Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis. EMBO J. 2018;37:e98684. doi: 10.15252/embj.201798684. PubMed DOI PMC

Carmen-Orozco, R. P. et al. Elevated nuclear TDP-43 induces constitutive exon skipping. Preprint at BioRxiv10.1101/2023.05.11.540291 (2023). PubMed PMC

Schlimgen AK, Helms JA, Vogel H, Perin MS. Neuronal pentraxin, a secreted protein with homology to acute phase proteins of the immune system. Neuron. 1995;14:519–526. doi: 10.1016/0896-6273(95)90308-9. PubMed DOI

Tsui CC, et al. Narp, a novel member of the pentraxin family, promotes neurite outgrowth and is dynamically regulated by neuronal activity. J. Neurosci. 1996;16:2463–2478. doi: 10.1523/JNEUROSCI.16-08-02463.1996. PubMed DOI PMC

Mackenzie IR, Neumann M. Reappraisal of TDP-43 pathology in FTLD-U subtypes. Acta Neuropathol. 2017;134:79–96. doi: 10.1007/s00401-017-1716-8. PubMed DOI

Falk A, et al. Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons. PLoS ONE. 2012;7:e29597. doi: 10.1371/journal.pone.0029597. PubMed DOI PMC

Held A, et al. iPSC motor neurons, but not other derived cell types, capture gene expression changes in postmortem sporadic ALS motor neurons. Cell Rep. 2023;42:113046. doi: 10.1016/j.celrep.2023.113046. PubMed DOI PMC

van der Ende EL, et al. Neuronal pentraxin 2: a synapse-derived CSF biomarker in genetic frontotemporal dementia. J. Neurol. Neurosurg. Psychiatry. 2020;91:612–621. doi: 10.1136/jnnp-2019-322493. PubMed DOI PMC

Xiao M-F, et al. NPTX2 and cognitive dysfunction in Alzheimer’s Disease. eLife. 2017;6:e23798. doi: 10.7554/eLife.23798. PubMed DOI PMC

Alzheimer’s Disease Neuroimaging Initiative. Neuronal Pentraxin 2 predicts medial temporal atrophy and memory decline across the Alzheimer’s disease spectrum. Brain Behav. Immun. 2016;58:201–208. doi: 10.1016/j.bbi.2016.07.148. PubMed DOI PMC

Xu D, et al. Narp and NP1 form heterocomplexes that function in developmental and activity-dependent synaptic plasticity. Neuron. 2003;39:513–528. doi: 10.1016/S0896-6273(03)00463-X. PubMed DOI

Mariga A, et al. Definition of a bidirectional activity-dependent pathway Involving BDNF and Narp. Cell Rep. 2015;13:1747–1756. doi: 10.1016/j.celrep.2015.10.064. PubMed DOI PMC

Lee S-J, et al. Presynaptic neuronal pentraxin receptor organizes excitatory and inhibitory synapses. J. Neurosci. 2017;37:1062–1080. doi: 10.1523/JNEUROSCI.2768-16.2016. PubMed DOI PMC

O’Brien R, et al. Synaptically targeted Narp plays an essential role in the aggregation of AMPA receptors at excitatory synapses in cultured spinal neurons. J. Neurosci. 2002;22:4487–4498. doi: 10.1523/JNEUROSCI.22-11-04487.2002. PubMed DOI PMC

Chang MC, et al. Narp regulates homeostatic scaling of excitatory synapses on parvalbumin-expressing interneurons. Nat. Neurosci. 2010;13:1090–1097. doi: 10.1038/nn.2621. PubMed DOI PMC

Wang Z, et al. Retrieval-driven hippocampal NPTX2 plasticity facilitates the extinction of cocaine-associated context memory. Biol. Psychiatry. 2020;87:979–991. doi: 10.1016/j.biopsych.2019.10.009. PubMed DOI

Rothstein JD. Excitotoxicity and neurodegeneration in amyotrophic lateral sclerosis. Clin. Neurosci. 1995;3:348–359. PubMed

Ilieva H, Polymenidou M, Cleveland DW. Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J. Cell Biol. 2009;187:761–772. doi: 10.1083/jcb.200908164. PubMed DOI PMC

Goel K, Ploski JE. RISC-y business: limitations of short hairpin RNA-mediated gene silencing in the brain and a discussion of CRISPR/Cas-based alternatives. Front. Mol. Neurosci. 2022;15:914430. doi: 10.3389/fnmol.2022.914430. PubMed DOI PMC

Hong H, et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature. 2009;460:1132–1135. doi: 10.1038/nature08235. PubMed DOI PMC

Okita K, et al. A more efficient method to generate integration-free human iPS cells. Nat. Methods. 2011;8:409–412. doi: 10.1038/nmeth.1591. PubMed DOI

Ling S-C, et al. ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc. Natl Acad. Sci. USA. 2010;107:13318–13323. doi: 10.1073/pnas.1008227107. PubMed DOI PMC

Avar M, et al. An arrayed genome-wide perturbation screen identifies the ribonucleoprotein Hnrnpk as rate-limiting for prion propagation. EMBO J. 2022;41:e112338. doi: 10.15252/embj.2022112338. PubMed DOI PMC

Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Berg S, et al. ilastik: interactive machine learning for (bio)image analysis. Nat. Methods. 2019;16:1226–1232. doi: 10.1038/s41592-019-0582-9. PubMed DOI

Pérez-Berlanga M, Laferrière F, Polymenidou M. SarkoSpin: a technique for biochemical isolation and characterization of pathological TDP-43 aggregates. Bio Protoc. 2019;9:e3424. PubMed PMC

Kerr JND, Greenberg D, Helmchen F. Imaging input and output of neocortical networks in vivo. Proc. Natl Acad. Sci. USA. 2005;102:14063–14068. doi: 10.1073/pnas.0506029102. PubMed DOI PMC

Stosiek C, Garaschuk O, Holthoff K, Konnerth A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl Acad. Sci. USA. 2003;100:7319–7324. doi: 10.1073/pnas.1232232100. PubMed DOI PMC

Thévenaz P, Ruttimann UE, Unser M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 1998;7:27–41. doi: 10.1109/83.650848. PubMed DOI

Pachitariu, M., Steinmetz, N., Kadir, S., Carandini, M. & Harris, K. D. Kilosort: realtime spike-sorting for extracellular electrophysiology with hundreds of channels. Preprint at BioRxiv10.1101/061481 (2016).

Germain P-L, Sonrel A, Robinson MD. pipeComp, a general framework for the evaluation of computational pipelines, reveals performant single cell RNA-seq preprocessing tools. Genome Biol. 2020;21:227. doi: 10.1186/s13059-020-02136-7. PubMed DOI PMC

McCarthy DJ, Campbell KR, Lun ATL, Wills QF. Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics. 2017;33:1179–1186. doi: 10.1093/bioinformatics/btw777. PubMed DOI PMC

Stuart T, et al. Comprehensive integration of single-cell data. Cell. 2019;177:1888–1902.e21. doi: 10.1016/j.cell.2019.05.031. PubMed DOI PMC

McInnes, L., Healy, J. & Melville, J. UMAP: uniform manifold approximation and projection for dimension reduction. J. Open Source Softw.3, 861 (2018).

Kowalczyk MS, et al. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells. Genome Res. 2015;25:1860–1872. doi: 10.1101/gr.192237.115. PubMed DOI PMC

Lun ATL, McCarthy DJ, Marioni JC. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. F1000Res. 2016;5:2122. PubMed PMC

Zhang JM, Kamath GM, Tse DN. Valid post-clustering differential analysis for single-cell RNA-seq. Cell Syst. 2019;9:383–392.e6. doi: 10.1016/j.cels.2019.07.012. PubMed DOI PMC

Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 2018;36:411–420. doi: 10.1038/nbt.4096. PubMed DOI PMC

Lütge A, et al. CellMixS: quantifying and visualizing batch effects in single-cell RNA-seq data. Life Sci. Alliance. 2021;4:e202001004. doi: 10.26508/lsa.202001004. PubMed DOI PMC

Trapnell C, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat. Biotechnol. 2014;32:381–386. doi: 10.1038/nbt.2859. PubMed DOI PMC

Cao J, et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature. 2019;566:496–502. doi: 10.1038/s41586-019-0969-x. PubMed DOI PMC

Crow M, Paul A, Ballouz S, Huang ZJ, Gillis J. Characterizing the replicability of cell types defined by single cell RNA-sequencing data using MetaNeighbor. Nat. Commun. 2018;9:884. doi: 10.1038/s41467-018-03282-0. PubMed DOI PMC

Orjuela S, Huang R, Hembach KM, Robinson MD, Soneson C. ARMOR: an automated reproducible modular workflow for preprocessing and differential analysis of RNA-seq data. G3. 2019;9:2089–2096. doi: 10.1534/g3.119.400185. PubMed DOI PMC

Ewels PA, et al. The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. 2020;38:276–278. doi: 10.1038/s41587-020-0439-x. PubMed DOI

Kuhn RM, Haussler D, Kent WJ. The UCSC genome browser and associated tools. Brief. Bioinformatics. 2013;14:144–161. doi: 10.1093/bib/bbs038. PubMed DOI PMC

Athar A, et al. ArrayExpress update—from bulk to single-cell expression data. Nucleic Acids Res. 2019;47:D711–D715. doi: 10.1093/nar/gky964. PubMed DOI PMC

Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139–140. doi: 10.1093/bioinformatics/btp616. PubMed DOI PMC

Lun ATL, Chen Y, Smyth GK. It’s DE-licious: a recipe for differential expression analyses of RNA-seq experiments using quasi-likelihood methods in edgeR. Methods Mol. Biol. 2016;1418:391–416. doi: 10.1007/978-1-4939-3578-9_19. PubMed DOI

Cunningham F, et al. Ensembl 2022. Nucleic Acids Res. 2022;50:D988–D995. doi: 10.1093/nar/gkab1049. PubMed DOI PMC

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal. 2011;17:10. doi: 10.14806/ej.17.1.200. DOI

Dobin A, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. PubMed DOI PMC

Shen S, et al. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-seq data. Proc. Natl Acad. Sci. USA. 2014;111:E5593–601. doi: 10.1073/pnas.1419161111. PubMed DOI PMC

Wickham H, et al. Welcome to the tidyverse. JOSS. 2019;4:1686. doi: 10.21105/joss.01686. DOI

Christmas MJ, et al. Evolutionary constraint and innovation across hundreds of placental mammals. Science. 2023;380:eabn3943. doi: 10.1126/science.abn3943. PubMed DOI PMC