{"paper_id":"8a3f8faf-3f5a-43c4-b84c-09f92bb32fc4","body_text":"1 \n\t\nMolecular\tsignatures\tof\tcortical\t\nexpansion\tin\tthe\thuman\tfetal\tbrain\t\n\t\nG.\tBall1,2\tS.\tOldham1\tV.\tKyriakopoulou3,4\tL.\tZ.\tJ.\tWilliams3,4\tV.\tKarolis3,5\tA.\tPrice3,4\tJ.\tHutter3,4\tM.L.\tSeal1,2\t\nA.\tAlexander-Bloch6,7,8,9\tJ.V.\tHajnal3,4\tA.D.\tEdwards3,4\tE.C.\tRobinson3,4\tJ.\tSeidlitz6,7,8,9\t\n1.\tDevelopmental\tImaging,\tMurdoch\tChildren’s\tResearch\tInstitute,\tMelbourne,\tAustralia\t\n2.\tDepartment\tof\tPaediatrics,\tUniversity\tof\tMelbourne,\tMelbourne,\tAustralia\t\n3.\tCentre\tfor\tthe\tDeveloping\tBrain,\tKing’s\tCollege\tLondon,\tLondon,\tUK\t\n4.\tSchool\tof\tBiomedical\tEngineering\t&\tImaging\tScience,\tKing’s\tCollege\tLondon,\tLondon,\tUK\t\n5.\tWellcome\tCentre\tfor\tIntegrative\tNeuroimaging,\tFMRIB,\tNuffield\tDepartment\tof\tClinical\t\nNeurosciences,\tUniversity\tof\tOxford,\tOxford,\tUK\t\n6.\tDepartment\tof\tChild\tand\tAdolescent\tPsychiatry\tand\tBehavioral\tSciences,\tThe\tChildren’s\tHospital\tof\t\nPhiladelphia,\tPhiladelphia,\tPA\t\n7.\tDepartment\tof\tPsychiatry,\tUniversity\tof\tPennsylvania,\tPhiladelphia,\tPA\t\n8.\tLifespan\tBrain\tInstitute,\tThe\tChildren’s\tHospital\tof\tPhiladelphia\tand\tPenn\tMedicine,\tPhiladelphia,\tPA\t\n9.\tInstitute\tof\tTranslational\tMedicine\tand\tTherapeutics,\tUniversity\tof\tPennsylvania,\tPhiladelphia,\tPA\t\n\t\n\t\nAbstract\t\nThe\tthird\ttrimester\tof\thuman\tgestation\tis\tcharacterised\tby\trapid\tincreases\tin\tbrain\tvolume\tand\t\ncortical\tsurface\tarea.\tA\tgrowing\tcatalogue\tof\tcells\tin\tthe\tprenatal\tbrain\thas\trevealed\tremarkable\t\nmolecular\tdiversity\tacross\tcortical\tareas.1,2\tDespite\tthis,\tlittle\tis\tknown\tabout\thow\tthis\ttranslates\tinto\t\nthe\tpatterns\tof\tdifferential\tcortical\texpansion\tobserved\tin\thumans\tduring\tthe\tlatter\tstages\tof\tgestation.\t\nHere\twe\tpresent\ta\tnew\tresource,\tμBrain,\tto\tfacilitate\tknowledge\ttranslation\tbetween\tmolecular\tand\t\nanatomical\tdescriptions\tof\tthe\tprenatal\tdeveloping\tbrain.\tBuilt\tusing\tgenerative\tartificial\tintelligence,\t\nμBrain\tis\ta\tthree-dimensional\tcellular-resolution\tdigital\tatlas\tcombining\tpublicly-available\tserial\t\nsections\tof\tthe\tpostmortem\thuman\tbrain\tat\t21\tweeks\tgestation3\twith\tbulk\ttissue\tmicroarray\tdata,\t\nsampled\tacross\t29\tcortical\tregions\tand\t5\ttransient\ttissue\tzones.4\tUsing\tμBrain,\twe\tevaluate\tthe\t\nmolecular\tsignatures\tof\tpreferentially-expanded\tcortical\tregions\tduring\thuman\tgestation,\tquantified\tin\t\nutero\tusing\tmagnetic\tresonance\timaging\t(MRI).\tWe\tfind\tthat\tdifferences\tin\tthe\trates\tof\texpansion\t\nacross\tcortical\tareas\tduring\tgestation\trespect\tanatomical\tand\tevolutionary\tboundaries\tbetween\t\ncortical\ttypes5\tand\tare\tfounded\tupon\textended\tperiods\tof\tupper-layer\tcortical\tneuron\tmigration\tthat\t\ncontinue\tbeyond\tmid-gestation.\tWe\tidentify\ta\tset\tof\tgenes\tthat\tare\tupregulated\tfrom\tmid-gestation\t\nand\thighly\texpressed\tin\trapidly\texpanding\tneocortex,\twhich\tare\timplicated\tin\tgenetic\tdisorders\twith\t\ncognitive\tsequelae.\tOur\tfindings\tdemonstrate\ta\tspatial\tcoupling\tbetween\tareal\tdifferences\tin\tthe\t\ntiming\tof\tneurogenesis\tand\trates\tof\texpansion\tacross\tthe\tneocortical\tsheet\tduring\tthe\tprenatal\tepoch.\t\nThe\tμBrain\tatlas\tis\tavailable\tfrom:\thttps://garedaba.github.io/micro-brain/\tand\tprovides\ta\tnew\ttool\tto\t\ncomprehensively\tmap\tearly\tbrain\tdevelopment\tacross\tdomains,\tmodel\tsystems\tand\tresolution\tscales.\t\n\t\nKeywords:\thuman\tbrain\tdevelopment;\tatlas;\ttranscriptomics;\tfetal;\tcortex\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n2 \nThe\thuman\tcortex\tis\ta\ttapestry\tof\tspecialised\tcortical\tareas\tsupporting\tdiverse\tand\tcomplex\tbehaviours,\teach\t\nidentifiable\ton\tthe\tbasis\tof\tdistinct\tpatterns\tof\tcyto-architecture,\tchemo-architecture,\tand\taxonal\tconnectivity.6–10\t\nDuring\tgestation,\twaves\tof\tneurons\tare\tgenerated\tfrom\tprogenitor\tcells\tlining\tthe\tcerebral\tventricles\tand\tmigrate\t\noutwards\talong\tsupporting\tradial\tglia\tto\tform\tthe\tlayers\tof\tthe\tcortex.11–13\tPrior\tto\tthe\tingress\tof\textrinsic\tconnections\t\nvia\tthe\tthalamus,14\tthe\tprogressive\tdifferentiation\tof\tcortical\tareas\tis\torchestrated\tby\ttranscription\tfactors\texpressed\t\nalong\tconcentration\tgradients\tand\ttranslated\tfrom\tthe\tventricular\tzone\t(VZ)\tto\tsecondary\tprogenitors\tof\tthe\t\nsubventricular\tzone\t(SVZ),\tthen\tonto\tneurons\tin\tthe\tcortical\tplate\t(CP),\tforming\tfunctional\tterritories.2,11,15–18\tThis\t\nprocess\tfollows\ta\tprecise\tspatiotemporal\tschema,11,18–22\tthe\ttraces\tof\twhich\textend\tfar\tbeyond\tthe\tnascent\tstages\tof\t\nneurogenesis\tand\tare\techoed\tin\tpatterns\tof\tcytoarchitecture,\taxonal\tconnectivity\tand\tfunction.23–29\t\t\nFocused\ton\tuncovering\tthe\tmechanisms\tthat\tgovern\tareal\tdifferentiation,\tstudies\thave\tbegun\tto\tcatalogue\tthe\tcellular\t\ndiversity\tof\tthe\tdeveloping\thuman\tcortex,\tand\tgenes\tthat\tencode\tit,\t\twith\tincreasing\tgranularity\tand\tscale.1,2,30–32\t\nRegional\tspecialisation\tof\tcell\ttypes\thas\tbeen\tobserved\tfrom\tearly\tin\tgestation,\twith\tdiversity\tof\tcortical\tgene\t\ntranscription\tmost\tevident\tin\tmid-\tto\tlate-gestation\tbut\tpersisting\tinto\tadulthood\tand\taligning\twith\tstructural\tand\t\nfunctional\torganisation\tof\tthe\tbrain.4,26,33–36\t\t\nThe\tthird\ttrimester\tof\thuman\tgestation\tis\tcharacterised\tby\trapid\tand\tsustained\tincreases\tin\tbrain\tvolume\tand\tcortical\t\nsurface\tarea.11,37,38\tDifferential\trates\tof\tareal\texpansion\tduring\thuman\tdevelopment\tmirror\tevolutionary\ttrends\tin\t\ncortical\tscaling\tand\tfunction39–44\twith\tpreferential\texpansion\tin\tareas\tvulnerable\tto\tdisruption\tin\t\nneurodevelopmental,45\tneurological46,\tgenetic47\tand\tpsychiatric48\tdisorders.\tJuxtaposed\thypotheses\timplicate\teither\tthe\t\nproduction\tof\tglia49,50,\tor\tneurons51–54\tfrom\tspecialised\tprogenitor\tpopulations\tof\tthe\touter\tSVZ,\tin\tthe\texpansion\tof\tthe\t\nprimate\tcortex.\tThus,\tthe\tdistribution\tof\tdistinct\tcell\tpopulations\tacross\tthe\tdeveloping\tcortex\tmay\tmediate\tareal\t\ndifferences\tin\texpansion\tand\tvulnerability\tto\tinsult18,33\tbut\twe\tcurrently\tdo\tnot\thave\ta\tclear\tunderstanding\tof\thow\tthis\t\nmolecular\tdiversity\tis\ttranslated\tinto\tcortical\torganisation\tin\thumans\tin\tvivo.\t\nμBrain:\tA\tthree-dimensional\tmicroscale\tatlas\tof\tthe\tfetal\tbrain\t\nTo\tbridge\tthis\tgap,\twe\tsought\tto\tconstruct\ta\t3D\tdigital\tatlas\tof\tthe\tdeveloping\tbrain\tat\tmicrometre\tscale\tusing\ta\tpublic\t\nresource\tof\t81\tserial\thistological\t2D\tsections\tof\ta\tprenatal\thuman\tbrain\tat\t21\tpostconceptional\tweeks\t(PCW).3,4\tSource\t\ndata\tincluded\tserial\tcoronal\tsections\t(20μm\tthickness)\tobtained\tfrom\tthe\tright\themisphere\tof\ta\tsingle\tprenatal\tbrain\t\nspecimen\t(21\tPCW;\tfemale),\tNissl-stained,\timaged\tat\t1\tmicron\tresolution\tand\tlabelled\twith\tdetailed\tanatomical\t\nannotations,\talongside\tinterleaved\tcoronal\tsections\tstained\twith\tin\tsitu\thybridisation\t(ISH)\tof\tn=41\tdevelopmental\tgene\t\nmarkers,\tas\treported\tby\tDing\tet\tal3\t(Figure\tS1;\tTable\tS1-S3;\tsee\tMethods).\tIn\tthis\tand\t3\tother\tspecimens\t(15,\t16\tand\t\n21\tPCW,\t2\tfemale),\tanatomical\tannotations\thad\tbeen\tused\tto\tguide\ta\tseries\tof\tlaser\tmicrodissections\t(LMD)\tacross\t\nmultiple\tcortical\tareas\tand\tlayers\tof\tthe\tcortical\tanlage\t(e.g.:\tcortical\tplate,\tsubplate,\tintermediate\tzone,\tventricular\t\nzone;\tTable\tS4)\tin\tthe\tleft\themisphere\tto\tmeasure\tregional\tgene\texpression\tvia\tRNA\tmicroarrays,\tas\tdescribed\tby\t\nMiller\tet\tal.4\t\tNissl-\tand\tISH-stained\tsections\twith\tcorresponding\tanatomical\tlabels\tand\tLMD\tarrays\twere\tmade\t\navailable\tas\tpart\tof\tthe\tBrainSpan\tDeveloping\tBrain\tAtlas\t[https://atlas.brain-map.org/atlas?atlas=3].\t\t\nArtefacts\tdue\tto\ttissue\tpreparation,\tsectioning\tand\tstaining\tprocedures\t(including\ttearing\tand\tfolding\tof\tsections)\tare\t\ncommon\tin\thistological\tdata\tand\tcan\tpresent\tdifficulties\tfor\tdownstream\tprocessing\tpipelines.55–58\tTo\tcorrect\tfor\ttissue\t\nartefacts\tpresent\tin\tthe\thistological\tdata,\twe\tdesigned\tan\tautomated\tdetect-and-repair\tpipeline\tfor\tNissl-stained\t\nsections\tbased\ton\tpix2pix,\ta\tGenerative\tAdversarial\tNetwork\t(GAN)59,60\t(Figure\t1a-d;\tSupplemental\tMethods).\tUsing\t\n256\t×\t256\tpixel\timage\tpatches\tdrawn\tfrom\t73/81\tlabeled\thistological\tsections\t(n=8\treserved\tfor\tmodel\ttesting)\twith\t\npaired\tanatomical\tlabels,\twe\ttrained\ta\tGAN\tmodel\tto\tproduce\tNissl-contrast\timages\tconditioned\ton\ta\tset\tof\t20\ttissue\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n3 \nlabels\t(Figure\t1a;\tTable\tS2).\tAfter\ttraining,\tthe\tmodel\twas\table\tto\tproduce\trealistic,\tNissl-stained\timage\tpatches\t\nmatched\ton\tcolour\thue\tand\tsaturation\tto\tthe\toriginal\tdata\tusing\ttissue\tannotations\talone\t(Figure\t1c).\tModel\t\nperformance\twas\trobust\tto\tdifferent\tparameter\tsettings\tand\tmodel\tarchitectures\t(Figure\tS2).\tUsing\tthe\ttrained\tmodel,\t\nwe\tgenerated\tsynthetic\tNissl-contrast\timage\tpredictions\tfrom\tanatomical\tannotations\tof\teach\tsection\tand\tidentified\t\nartefacts\tin\tthe\thistological\tdata\tbased\ton\tdeviations\tin\tpixel\thue\tand\tsaturation\tfrom\tthe\tmodel\tprediction.\tOutlier\t\npixels\twere\treplaced\twith\tmodel\tpredictions\tusing\tPoisson\timage\tediting61\t(Figure\t1d)\tresulting\tin\tn=79\t(2\texcluded\t\ndue\tto\textensive\tmissing\ttissue)\tcomplete\thistological\tsections\t(Figure\tS1;\tTable\tS1).\t\t\nHistological\tatlases\tof\tthe\tcerebral\tcortex6,10\thave\tproven\tinvaluable\tfor\tunderstanding\thuman\tbrain\torganisation\tbut\t\nare\tlimited\tby\tthe\tloss\tof\tspatial\tinformation\tinherent\tto\t2D\trepresentations\tof\t3D\tstructures.\tReconstructions\tof\t3D\t\nbrain\tvolumes\tfrom\tserial\ttissue\tsections\tof\tpost\tmortem\ttissue\tallow\tthe\texamination\tof\tintact\tbrain\tanatomy\tat\ta\tscale\t\ninaccessible\tto\tcurrent\tneuroimaging\ttechnologies.62\tWe\tcombined\trepaired\ttissue\tsections\tinto\ta\t3D\tvolume\tof\tthe\t\nright\themisphere\tusing\titerative\taffine\timage\tregistration\tconstrained\tby\ta\ttissue\tshape\treference\tderived\tfrom\tfetal\t\nMRI\t(Figure\tS3;\tSupplemental\tMethods),63\tfollowed\tby\tnonlinear\talignment\tto\taccount\tfor\twarping\tbetween\tadjacent\t\nsections.\tUsing\tthe\taligned\tdata,\twe\tgenerated\ta\t3D\tvolume\tresampled\tto\tvoxel\tresolution\t150\t×\t150\t×\t150𝜇m\twith\t\ndimension\t189\t×\t424\t×\t483\tvoxels\t(28.35\t×\t63.60\t×\t72.45mm)\t(μBrain;\tsee\tMethods;\tFigure\t1e,\tFigure\tS4a-b).\t\nFollowing\treconstruction,\twe\tbenchmarked\tthe\tsize\tof\tthe\treconstructed\tμBrain\tvolume\tagainst\tstandard\tfetal\tgrowth\t\nmetrics\tfor\ta\t23\tweek\t(gestational\tage;\tGA,\tequivalent\tto\t21\tPCW)\tfetus\t(μBrain\tlength\t=\t62.7mm,\t23\tweek\tGA\toccipital-\nfrontal\tdiameter\tmedian\t[5th,\t95th\tcentile]\t=\t73.3\tmm\t[68.2,\t78.5]),64\tand\tcompared\ttissue\tvolume\testimates\tbased\ton\t\nreconstructed\tanatomical\tlabels\t(parenchymal\tvolume\t=\t25.8ml,\tright\themisphere)\tto\tpreviously\treported\t3D\tMRI-\nderived\tfetal\tbrain\tvolumes\t(supratentorial\tvolume\t[both\themispheres]\tat\t23\tweek\tGA\t=\t60.26ml).65\tAdapting\tprotocols\t\nfrom\tneuroimaging\tanalysis,\twe\textracted\tthe\tinner\tand\touter\tsurfaces\tof\tthe\tcortical\tplate\tand\tprojected\ta\tset\tof\t29\t\ncortical\tarea\tlabels\tderived\tfrom\tthe\thistological\ttissue\tannotations\t(Table\tS2)\tonto\tthe\tsurface\tvertices\tto\tform\tthe\t\nμBrain\tcortical\tatlas\t(Figure\t1f).\tThe\tμBrain\tcortical\tatlas\trepresents\ta\tnew\tparcellation\tof\tthe\tdeveloping\tbrain\tdefined\t\naccording\tto\tthe\thierarchical\tontology\tof\tthe\treference\tannotations\tand\tmatched\tto\tcorresponding\tLMD\tmicroarray\t\ndata\t(Table\tS2,\tS4;\tFigure\t4c-d).\t\t\nIn\taddition\tto\tthe\twhole\tbrain\tvolume\tand\tcortical\tatlas,\twe\tcreated\tpartial\t3D\treconstructions\tof\tISH\tstaining\tfor\t41\t\ngenes\t(see\tMethods;\tFigure\t1g).\tBased\ton\tan\taverage\t41\ttissue\tsections\tper\tgene\t(Table\tS3),\tsemi-quantitative\tmaps\t\nof\tgene\texpression\trevealed\tthe\ttissue-\tand\tregion-specific\tdistributions\tof\tseveral\tgenes,\tincluding\tcaudal\tenrichment\t\nof\tthe\ttranscription\tfactor\tEOMES\tin\tthe\tsubventricular\tzone,15\tand\tmarkers\tof\tneuronal\tmigration\t(DCX66)\tand\tsynaptic\t\ntransmission\t(GRIK267),\tin\tthe\tcortical\tplate\t(Figure\t1g;\tFigure\tS5)\t\nExisting\thistological\tbrain\tatlases,\tincluding\tthose\tof\tthe\tadult\thuman,62,68,69\tmouse,70,71\tand\tmacaque72\tbrains\tfacilitate\t\nintegration\twith\tother\tdata\tmodalities,\tincluding\tneuroimaging,\tand\tare\tamenable\tto\tadvanced\tcomputational\timage\t\nanalysis\tmethods\tto\textract\tquantitative\tmeasures\tof\tneuroanatomy\tacross\tmultiple\tscales.73,74\tBuilding\tupon\texisting\t\nresources,3,4\twe\thave\tcreated\tthe\tμBrain\tatlas\t(Figure\t1;\tFigure\tS4),\ta\tnew\tand\tfreely-available\t3D\tvolumetric\tmodel\tof\t\nthe\t21\tPCW\tfetal\tbrain\tat\t150𝜇m\tresolution,\taccompanied\tby\ta\tset\tof\tn=20\tcerebral\ttissue\tlabels\t(Figure\tS4a-b);\t\nsurface\tmodels\tof\tthe\tcortical\tplate\tsurface\tand\tcortical\tplate/subplate\tinterface\twith\tn=29\tcortical\tarea\tlabels\t(Figure\t\n4c)\tand\tn=41\tpartial\treconstructions\tof\tISH\texpression\tdata\t(Figure\t5).\tCortical\tareas\tare\tmatched\tto\tnormalised\tgene\t\nexpression\tdata\tfrom\tcorresponding\tLMD\tmicroarrays\t(Table\tS4;\tFigure\tS4d)\tacross\tmultiple\ttissue\tzones\tin\tthree\t\nadditional\tprenatal\tspecimens\t(total\tn=4)\tproviding\ta\t3D\tanatomical\tcoordinate\tspace\tto\tfacilitate\tintegrated\timaging-\ntranscriptomic\tanalyses\tof\tthe\tdeveloping\tbrain.\tBelow,\twe\tuse\tthe\tμBrain\tatlas\tto\tevaluate\tthe\tmolecular\tand\tcellular\t\ncorrelates\tof\tcortical\texpansion\tin\tthe\tthird\ttrimester\tof\thuman\tgestation.\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n4 \n\t\nFigure\t1:\tGeneration\tof\ta\t3D\tanatomical\tatlas\tof\tthe\tmid -gestation\tfetal\tbrain.\ta.\tPaired\thistological\tsections\tand\tsimplified\tanatomical\t\nannotations\twere\tdivided\tinto\t256\t ×\t256\trandom\tpatches\t(n=1000)\tfor\tmodel\ttraining.\tPatches\twere\tquality\tchecked\tprior\tto\tselection\tto\tensure\t\ngood\toverlap\tbetween\tlabels\tand\tanatomy\tand\tno\ttissue\tdamage.\t b.\tPix2pix\tmodel\tarchitecture\tshowing\ta\tU -Net\tgenerator\tcoupled\twith\ta\tPatchGAN\t\ndiscriminator.\tBox\tsizes\trepresent\timage\twidth,\theight\tand\tnumber\tof\tfilters/channels\t(depth)\tat\teach\tlayer.\tFilters\tand\tdime nsions\tof\teach\tlayer\tare\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n5 \nshown\tbelow.\tc.\tModel\tperformance\twas\tevaluated\ton\ta\tset\tof\tsections\tthat\twere\tnot\tincluded\tin\tthe\ttraining\tdataset.\t Checkerboard\tocclusions\tare\t\nshown\twith\tthe\toriginal\tsection,\toccluded\tpatch\tpredictions\tare\tshown\tusing\tthe\ttrained\tmodel\tafter\ta\tgiven\tnumber\tof\titerati ons.\td.\tThe\ttrained\t\nmodel\twas\tused\tto\treplace\tRGB\tvalues\tof\toutlying\tpixels\twith\tsynthetic\testimates.\tTop\trow:\toriginal\tsections\tspaced\t throughout\tthe\tcerebral \t\nhemisphere\twith\tautomatically\tidentified\toutlier\tpixels\toutlined\tin\tgrey.\tBottom\trow,\trepaired\tsections.\t e.\tRepaired\tsections\twere\taligned\tvia\tlinear,\t\naffine\tand\titerative\tnonlinear\tregistrations\t(see\t Methods)\tto\tcreate\ta\t3D\tvolume\twith\tfinal\tisotropic\tresolution\tof\t150um.\t Right:\tCut-planes\tillustrate\t\ninternal\tstructures\tafter\teach\tstage\tof\treconstruction.\tThe\treconstructed\ttissue\tlabel\tvolume\tis\tshown\tin\t Figure\tS4.\tf.\tThe\touter\t(pial)\tand\tinner\t\n(subplate)\tcortical\tplate\tboundaries\twere\textracted\tas\tsurface\ttessellations.\t The\tμBrain\tcortical\tlabels\twere\tprojected\tonto\tthe\tsurface\tvertices\tto\t\nform\tthe\tfinal\tcortical\tatlas\t(see\tFigure\tS4).\tCortical\tareas\tcorrespond\tto\tmatched\tLMD\tmicroarray\tdata\t( Table\tS4;\tFigure\tS4d).\tg.\tPartial\t\nreconstructions\tof\tEOMES,\tFOXP1\tand\tGRIK2\tISH\tdata.\tISH\tstained\tsections\twere\tregistered\tto\tnearest\tNissl -stained\tsections\tand\taligned\tto\tthe\tμBrain\t\nvolume.\tTop\trow:\tselected\taxial\tand\tcoronal\tsections\tof\tth e\tμBrain\tvolume\tand\tcorresponding\ttissue\tlabels\twith\tISH\texpression\tof\tthree\t\ndevelopmental\tgenes:\tEOMES,\tFOXP1\tand\tGRIK2\toverlaid.\tExpression\tintensity\twas\tderived\tfrom\tfalse -colour,\tsemi-quantitative\tmaps\tof\tgene\t\nexpression.\tBottom\trow:\taverage\texpression\tintensity\twithin\teach\ttissue\tor\tbrain\tstructure\tbased\to n\tμBrain\ttissue\tlabels.\tAverages\twere\tcalculated\t\nonly\twithin\tsections\twhere\tISH\twas\tavailable\tfor\teach\tgene. \t\n\t\nTissue-\tand\tregion-specific\tgene\texpression\tin\tthe\tmid-gestation\tbrain\t\nWe\tsought\tto\tcharacterise\tpatterns\tof\tgene\texpression\tin\tthe\tmid-gestation\tbrain\tand\tidentify\tdevelopmental\tand\t\nregion-specific\tgenes\twith\tputative\troles\tin\tcortical\texpansion.\tTo\tdo\tso,\twe\tused\tpublicly-available\tmicroarray\tdata\t\nfrom\tfour\tprenatal\tbrain\tspecimens\taged\t15\tto\t21\tPCW.4\t\tMicroarray\tprobe\tannotations\twere\tupdated\tand\ttissue\t\nsamples\tmatched\tto\tthe\tμBrain\tatlas\t(Table\tS4)\tyielding\texpression\tdata\tof\t8771\tgenes\tsampled\tfrom\tbetween\t18\tand\t\n27\tbrain\tregions\tand\tacross\t5\ttissue\tzones\tfor\teach\tspecimen\t(see\tMethods;\tFigure\tS4d).\tApplying\tPCA\tto\tgene\t\nexpression\tprofiles,\twe\tfound\tthat\ttissue\tsamples\twere\tprimarily\tseparated\taccording\tto\tlocation\tin\tmitotic\t(VZ,\tSVZ)\tor\t\npost-mitotic\ttissue\tzones,\trather\tthan\tacross\tregions\t(Figure\t2a)4\t–\ta\tpattern\tthat\twas\treplicated\tacross\tall\tspecimens\t\nwhen\tanalysed\tseparately\t(Figure\tS6).\tFocusing\ton\texpression\tprofiles\twithin\teach\ttissue\tzone,\tsamples\tclustered\t\naccording\tto\tmaturity\t(Figure\t2a;\tFigure\tS7)\twith\tdevelopmental\tchanges\tin\tgene\texpression\tmost\tsimilar\tacross\t\nadjacent\tmitotic\t(CP\tand\tSP,\tr=\t0.67)\tand\tpost-mitotic\tzones\t(SVZ\tand\tVZ,\tr=0.43;\tFigure\tS8).\tIn\tline\twith\tevidence\tof\ta\t\ntransition\tin\tVZ\tcell\tfate\taround\tmid-gestation,75\twe\tobserved\tincreased\texpression\tof\tgenes\tenriched\tin\tpost-mitotic\t\nexcitatory\tneurons\tand\tinterneurons\t(e.g.:\tGRIK1-3;\tGLRA2;\tSCN3B)\tbetween\t15\tand\t21\tPCW.1\tIn\tthe\tSP\tand\tCP,\tthis\t\ntransition\tcoincided\twith\tan\tincrease\tin\tgenes\texpressed\tby\tradial\tglia\t(BMP7;\tSOX3)\tand\toligodendrocyte\tprecursor\t\ncells\t(OPCs;\tCA10)\twith\ta\ttransitory\tdecrease\tin\tmicroglia-enriched\tgenes\tin\tthe\tCP\t(GPR34;\tTREM2)76\t(Figure\t2b;\t\nTable\tS5).\t\t\nAcross\tall\ttissue\tsamples,\twe\ttested\tfor\tdifferences\tin\tgene\texpression\tacross\tzones\t(CP,\tSP,\tetc.),\tregions\t(motor,\t\nsensory,\tetc)\tand\ttimepoints\t(early\tvs\tmid-gestation).\tThis\tresulted\tin\ta\tsubset\tof\tn=2145\t(24.5%)\tgenes\twith\t\ndifferential\texpression\tacross\tall\tthree\tfactors,\ttermed\tZone-Region-Tissue\t(ZRT)\tgenes\t(p<0.01\tafter\tFDR\tcorrection;\t\nFigure\t2c;\tTable\tS6).\tWe\treasoned\tthat\tthis\tsubset,\tcharacterised\tby\tgenes\twith\tdynamic\tregional\tand\ttemporal\t\nexpression\tin\tmid-gestation,\twould\tbe\tassociated\twith\tdifferential\trates\tof\tcortical\texpansion\tduring\tdevelopment.\tTo\t\nsupport\tthis\tline\tof\treasoning,\twe\tfound\tthat\tthe\tZRT\tcluster\twas\tenriched\tfor\tgenes\tupregulated\tin\tthe\tthird\t\ntrimester77\t(enrichment\tratio\t=\t1.89,\thypergeometric\ttest\tphypergeom<0.0001)\tand\thighly\texpressed\tin\tadolescent\tand\t\nadult\tbrain\ttissue,\tcompared\tto\tnon-ZRT\tgenes\t(Figure\tS9).\tZRT\tgenes\tincluded\tseveral\thuman\ttranscription\tfactors\t\n(e.g.:\tEGR1,\tJUNB,\tZNF536)78\tand\twere\tsignificantly\tenriched\tin\tradial\tglia\t(SOX2,\tHES5,\tphypergeom=0.03176),\tOPCs\t\n(OLIG1,\tPDGFRA;\tphypergeom=0.0424)\tand\tmigrating\tinterneurons\t(CALB2,\tCNR1;\tphypergeom=0.0009;\tFigure\t2d;\tTable\t\nS7).2\tWe\tobserved\thighest\tZRT\texpression\tin\tthe\tsubplate,\twith\tincreasing\texpression\tof\tZRT\tgenes\tin\tpostmitotic\t\nzones\t(CP,\tSP\tand\tIZ)\tcompared\tto\tthe\tSVZ\tand\tVZ,\tbetween\t15\tand\t21\tPCW\t(Figure\t2e).\tExamining\tZRT\tgene\t\nannotations\trevealed\tenrichment\tof\tcritical\tneurodevelopmental\tfunctions\tincluding\tcell-cell\tadhesion\t(GO:\t0098742;\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n6 \nCHD1,\tEFNA5,\tNLGN1,\tNRXN1;\tpFDR<0.0001,\tbackground\tset\t=\t8771\tgenes),\tforebrain\tdevelopment\t(GO:\t0030900;\t\nCASP3,\tCNTN2,\tDLX2,\tFOXP2,\tNEUROD6;\tpFDR=0.026)\tand\tneuron\tprojection\tguidance\t(GO:\t0097485;\tEFNA2,\tEFNA5;\t\npFDR\t=\t0.0034)\t(Table\tS8).\tThe\tZRT\tgeneset\twas\tadditionally\tenriched\tfor\thigh-confidence\tASD-linked\tgenes\t(n\t=\t43,\t\nphypergeom\t=\t0.034)79\tincluding\tSCL6A1,\tCACNA1C\tand\tCHD7\tand\tpathogenic\tvariants\tin\t161\tZRT\t(7.5%)\tgenes\thave\tbeen\t\nlinked\tto\tneurodevelopmental\tand\tcognitive\tphenotypes\tand\tbrain\tmalformations80\tincluding\tMAGEL2\t(Schaaf-Yang\t\nsyndrome81),\tAFF2\t(Fragile-X-E82)\tand\tADGRG1\t(polymicrogyria83)\t(Table\tS9)\t\n\t\nFigure\t2:\tRegional\tgene\texpression\tin\tthe\tmid -gestation\tfetal\tbrain.\ta.\tPCA\tof\tLMD\tmicroarray\tdata\t(n=8771\tgenes)\tin\tfour\tprenatal\tbrain\t\nspecimens\taged\t15\tPCW\tto\t21\tPCW.\tAll\ttissue\tsamples\tare\tshown\t(left)\tcoloured\tby\ttissue\tzones\t(main)\tand\tspecimen\t(inset).\tPC A\twas\tapplied\tto\tall\t\nsamples\tin\teach\ttissue\tzone\tseparately\t(righ t).\tSamples\tare\tcoloured\tby\tspecimen\tand\tcluster\tby\tage.\t b.\tPC1\twas\tassociated\twith\tage -related\tchange\t\nin\tall\ttissues\tand\tcorrelated\tbetween\tneighbouring\tzones.\tPlots\tshow\tmean\tgene\texpression\tat\t21\tPCW\t(averaged\tover\tspecimen\ta nd\tregion)\tagainst\t\nfold\tchange\tin\tgene\texpression\tbetween\t15/16\tPCW\tand\t21\tPCW\tfor\ttwo \ttissue\tzones\t(cortical\tplate,\ttop\tand\tventricular\tzone,\tbottom).\tGenes\twith\ta\t\nlog2(fold\tchange)\t>\t0.3\tare\tshown\tin\tgreen\t(< -0.3\tin\tblue).\tRepresentative\tgenes\tare\thighlighted.\t c.\tNumber\tof\tgenes\twith\tdifferential\texpression\tover\t\ntissue\tzones\t(ZONE),\tcortical\tregion\t(REGION)\tor\ttimepoint\t(TIME).\tVenn\tdiagram\tshows\toverlap\tof\tgene\tsets.\tIn\ttotal,\tn=2145\t were\tdifferentially\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n7 \nexpressed\tacross\tzone,\tregion\tand\ttime\t(ZRT\tgenes).\t d.\tenrichment\tof\tZRT\tgenes\tin\tcell\ttypes\tpreviously\tidentified\tin\tthe\tmid -gestation\tfetal\tbrain\t\n(left).2\tUMAP\tprojection\tof\tcell\ttypes\t showing\tenriched\tclusters\tof\tOPCs\tand\tradial\tglia.\t Inset:\tUMAP\tprojection\tcoloured\tby\tcell\ttype.\t e.\tZRT\tgene\t\nexpression\tover\ttime\tand\tregion.\tWedge\tplots\t(left)\tshow\tthe\tpattern\tof\texpression\tof\tZRT\tgenes\tthat\tdecrease\t(left)\tor\tincre ase\t(right)\tbetween\t15\t\nand\t21\tPCW.\tRows\tindicate\ttissue\tzones\tand\tcolumns\tindicate\tcortical\tregions\tordered\tfrom\tanterior \tto\tposterior\tpoles.\tBoxes\tare\tcoloured\tby\tchange\t\nin\tgene\texpression\tover\ttime\t( 𝛥\texpression).\tRight:\tbar\tcharts\tshow\tmean\tchange\tin\tgene\texpression\tfor\tdecreasing\t(top)\tand\tincreasing\t(bottom)\t\nZRT\tgenes\taveraged\twithin\ttissue\tzones. \t\n\t\nRegional\tdifferences\tin\tthe\trate\tof\tcortical\texpansion\tin\tutero\tduring\tthe\tthird\ttrimester\t\nWe\thypothesised\tthat\tthe\tdynamic\ttemporal\tand\tregional\tpatterning\tof\tZRT\tgenes\tacross\ttissue\tzones\tsupport\t\ndifferential\trates\tof\tareal\texpansion\tacross\tthe\tcortex.\tTo\ttest\tthis,\twe\tacquired\tn=240\tmotion-corrected\tfetal\tbrain\tMRI\t\nscans\tfrom\t229\tfetuses\taged\tbetween\t21+1\tand\t38+2\tgestational\tweeks+days\tas\tpart\tof\tthe\tDeveloping\tHuman\t\nConnectome\tProject\t(dHCP).84\tVolumetric\tT2-weighted\tscans\twere\tautomatically\treconstructed\tto\t0.5mm\tisotropic\t\nvoxel\tresolution85,86;\tthen\ttissue\tsegmentations\twere\tinitially\textracted\tusing\tneonatal\tprotocols87\t,\tfollowed\tby\t\nextensive\tmanual\tediting\tto\tensure\taccuracy\t(Methods).\tManually-corrected\tsegmentations\twere\tthen\tused\tto\tgenerate\t\ncortical\tsurface\treconstructions\t(Figure\t3a).88\tFor\tanalysis,\tindividual\tcortical\tsurfaces\twere\taligned\tto\ta\tfetal\t\nspatiotemporal\tatlas\tusing\ta\tnonlinear,\tbiomechanically-constrained\tsurface\tregistration\t(Multimodal\tSurface\tMatching\t\n[MSM];\tFigure\t3a-c).89–92\tAt\teach\tstage,\toutputs\twere\tvisually\tquality-checked\tand\tany\tfailures\tremoved.\tIn\ttotal,\tdata\t\nfrom\tn=195\tscans\tin\t190\tfetuses\t(gestational\tage:\t21+1\t-\t38+2\tweeks;\t88\tfemale)\twere\tincluded\tin\tthe\tanalysis\t(Figure\t\nS10).\t\t\nAs\texpected,\ttotal\tcortical\tsurface\tarea\tincreased\texponentially\tbetween\t21-\tand\t38-weeks\tgestation\t(βage=0.054,\t\np<0.001;\tFigure\t3c-d).93–95\tWhile\tcortical\tsurface\tarea\twas\tmoderately\tgreater\tin\tmales\tcompared\tto\tfemales\t\n(βmale=0.011,\tp=0.002),\tthis\trelationship\tdid\tnot\tchange\twith\tage\t(p=0.946).\tAt\teach\tvertex\tin\tthe\tcortical\tsurface\tmesh\t\n(n=30,248,\texcluding\tmidline\tregions),\twe\tmodelled\tareal\texpansion\twith\trespect\tto\ttotal\tsurface\tarea\tusing\tlog-log\t\nregression\t(see\tMethods;\tFigure\t3e).42\tHyperallometric\texpansion,\toccurring\tat\ta\trate\tfaster\tthan\tthe\tcortical\tsurface\t\nas\ta\twhole,\twas\tobserved\tacross\tthe\tlateral\tneocortical\tsurface\tencompassing\tthe\tfronto-parietal\toperculum\tand\t\n(granular)\tinsula,\tprimary\tmotor\tand\tsensory\tcortex\tas\twell\tas\tdorsal\tparietal\tand\tfrontal\tcortices,\tconfirming\tprevious\t\nobservations\tbased\ton\tfetal\tMRI\tand\tscans\tof\tpreterm-born\tinfants\t(Figure\t3c,\te).95–98\tIn\tline\twith\tproposed\tmodels\tof\t\ncortical\tevolution\tand\texpansion,5,9\tslower\trates\tof\tgrowth\twere\tobserved\tin\tmedial\tallocortex\t(including\tentorhinal,\t\npaleocortex\tand\tparahippocampal\tcortex)\tand\tthe\tcingulate\tcortex\t(Figure\t3e).\tThe\tinclusion\tof\tsex\tand\tsex:age\t\ninteraction\teffects\tin\tthe\tscaling\tmodel\tdid\tnot\taffect\testimated\tvertex\tscaling\tcoefficients\t(r\t=\t0.996).\t\tWe\tconfirmed\t\nthat\testimates\tof\tcortical\texpansion\tfrom\tcross-sectional\tanalysis\taligned\tclosely\tto\tlongitudinal\tobservations\tfrom\ta\t\nsingle\tfetus\tscanned\tthree\ttimes\tduring\tgestation\t(Figure\tS11).\t\nWe\tcalculated\tthe\tnon-parametric\tcorrelation\t(Kendall’s\t𝜏)\tbetween\tregional\testimates\tof\tZRT\tgene\texpression\tin\tthe\t\ncortical\tplate\tand\tsubjacent\ttissue\tzones\tand\taverage\tallometric\tscaling\tin\teach\tof\tcortical\tareas\tdefined\tby\tthe\tμBrain\t\natlas\t(Figure\t3e).\tIn\ttotal,\tacross\tboth\tearly\tand\tmid-gestation\ttimepoints,\texpression\tof\t433/2145\t(20.1%)\tZRT\tgenes\t\nwas\tspatially\tcorrelated\twith\tareal\texpansion\tduring\tgestation\tin\tat\tleast\tone\ttissue\tzone\t(ZRTscaling;\tn=542\tsignificant\t\nassociations,\tpFDR\t<\t0.01)\t(Table\tS9,\tS10).\tAssociations\twith\tareal\tscaling\twere\tsignificantly\tmore\tcommon\tin\tZRT\t\ngenes\tthan\tin\tnon-ZRT\tgenes\t(ZRT:\t20.1%,\tnon-ZRT:\t8.3%;\todds\tratio=2.78,\tp<0.0001)\twith\tmost\tsignificant\tZRTscaling\t\nassociations\t(414/542)\tlocalised\tto\tthe\tCP\t(Figure\t3f;\tTable\tS10).\tZRTscaling\tgenes\tin\tthe\tCP\tincluded\tknown\tmolecular\t\ncorrelates\tof\tareal\tidentity\t(EFNA5,99\tGLI3,100\tFGFR2101)\tand\taxonal\tguidance\t(SLIT1,\tROBO3,\tSRGAP1).102\t\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n8 \nDifferential\texpression\tof\tZRTscaling\tgenes\tlargely\tcaptured\tdifferences\tbetween\tpost-mitotic\tallocortex\tand\tneocortex,\t\nreflecting\topposing\tallometric\tscaling\tacross\tphylogenetic\tcortical\ttypes\t(Figure\t3e).\tWe\tfound\tevidence\tat\t15\tPCW,\tbut\t\nnot\tat\t21\tPCW,\tthat\tgenes\twith\thigher\texpression\tin\tslower-expanding\tallocortex\tand\tperi-allocortex,\twere\tsignificantly\t\nenriched\tin\tearly-born\tCajal-Retzius\tneurons\t(e.g.:\tCALB2;\toverlap=17,\tenrichment=1.72,\tphypergeom=0.021),2\tcells\tthat\t\noriginate\tfrom\tthe\tpallial-subpallial\tboundary\tand\tcortical\them\tand\tmigrate\ttangentially\tacross\tthe\tdeveloping\t\nneocortex\tin\tearly\tgestation.103,104\tZRTscaling\tgenes\tinvolved\tin\tNotch\tsignalling\t(NOTCH2NLR,\tJAG1)105\tand\tothers\tcritical\t\nfor\thippocampal\tdendritic\tdevelopment\t(LRIG1)106\twere\talso\texpressed\thighly\tin\tallocortical\tregions\t(Table\tS10).\tIn\t\ncontrast,\tZRTscaling\tgenes\texpressed\tin\tthe\tpreferentially\texpanded\tneocortex\twere\tenriched\tin\tprogenitor\tcells\tat\t15\t\nPCW\t(FBXO32,\tHES6;\tIPC\tenrichment\t=\t1.51,\tphypergeom\t=\t0.027),\tand\tgeneral\tmarkers\tof\tdeep\tlayer\tneurons\tat\tboth\t\ntimepoints\t(NEUROD6,\tSYT6;\t15\tPCW:\tNeuron\tenrichment\t=\t1.53,\tphypergeom\t=\t0.004;\t21\tPCW:\tenrichment\t=\t1.51,\t\nphypergeom\t=\t0.007).\tWhile\tbasic\tcell\ttypes\tare\tgenerally\tconserved\tacross\tcortical\tareas,107\tprevious\tevidence\thas\tshown\t\nthat\tregional\tidentity\tis\timprinted\tduring\tcell\tdifferentiation,\twith\tareal\tsignatures\tmost\tapparent\tin\tpost-mitotic\tcell\t\ntypes\tbut\tpervasive\teven\tat\tearly\tstages\tof\tdevelopment\tacross\tmajor\tbrain\tstructures.2,15,31\tIn\tline\twith\tthis,\twe\tfound\t\nopposing\tenrichment\tof\tpostmitotic\tneuronal\tmarkers\tspecific\tto\tallocortex\tand\tneocortex\tin\thypoallometric\tand\t\nhyperallometric\tZRTscaling\tgenes,\trespectively\t(Figure\t3g).\t\n\t\nFigure\t3:\tPreferential\tcortical\texpansion\tduring\tthe\tthird\ttrimester.\ta.\t n=195\tfetal\tMRI\tscans\twere\tacquired\tduring\tthe\tthird\ttrimester\tof\t\npregnancy.\tT2-weighted\t(T2W)\tscans\twere\treconstructed\tusing\ta\tmotion -robust\tprocessing\tpipeline\tand\tused\tto\tgenerate\ttessellated\tcortical\tsurface\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n9 \nrepresentations\tthat\twere\taligned\tto\tthe\tdHCP\tfetal\tsurface\ttemplate\t b.\tμBrain\tcortical\tlabels\tprojected\tonto\tdHCP\tfetal\ttemplate\tsurfaces\tfrom\t21\tto\t\n36\tweeks\tgestation\tusing\tnonlinear\tsurface\tregistration .\tSurfaces\tare\tscaled\tto\tthe\tsame\tsize\tfor\tvisualisation.\t c.\tFor\teach\ttimepoint,\tweighted\taverage\t\nvertex\tarea\tmaps\tare\tdisplayed\ton\tthe\trespective\tsurface\ttemplates.\tFetal\tcortical\tarea\tmaps\twere\tcalculated\tfrom\tindividual, \tco-registered\tand\t\nresampled\tfetal\tsurfaces\tusing\ta\tGaussian\tkernel\t(sigma\t=\t1\tweek).\t d.\ttotal\tcortical\tsurface\tarea\tcalculated\tacross\tall\tsurface\tvertices\t(excluding\tthe\t\nmidline)\tas\ta\tfunction\tof\tgestational\tage\tat\tscan.\t e.\tLeft:\tModels\tof\tallometric\tscaling \twere\tcalculated\tfor\teach\tvertex,\tmodelling\tlog 10(vertex\tarea)\tas\ta\t\nfunction\tof\tlog10(total\tarea)(top).\tIn\tthis\tframework,\t 𝛽>1\tindicates\thyperallometric\tgrowth\t(a\trelative\texpansion\tfaster\tthan\tthe\tglobal\trate).\tNote\t\nthat\ta\tfaster\tgrowth\trate\tdoes\tnot\tnecessarily\tequate\tto\tgreater\ttotal\tarea\tat\tany\tgiven\ttime\t(bottom).\tMiddle: \tHyperallometric\tscaling\twith\trespect\tto\t\ntotal\tcortical\tsurface\tarea\t( 𝜷 > 𝟏)\tplotted\ton\tthe\t36w\ttemplate\tsurface\trepresenting\tpreferential\tcortical\texpansion\tduring\tdevelopment.\tRight:\t\nDistribution\tof\tscaling\tcoefficients\tfor\tall\tvertices\tin\teac h\tμBrain\tlabel\tin\ta,\tordered\tby\tmean\tscaling.\t f.\tRight:\tIn\ttotal,\texpression\tof\t433\tZRT\tgenes\t\nwere\tcorrelated\twith\tareal\tscaling\tin\tgestation.\tLeft:\tSignificant\tassociations\t(Kendall’s\t 𝜏,\tpFDR<0.01)\twere\tobserved\tacross\tboth\tearly\t(15/16\tPCW,\t\nn=2)\tand\tmid-gestation\t(21\tPCW,\tn=2)\ttimepoints\tand\tin\tall\ttissue\tzones.\t g.\tenrichment\tof\thypoallometric\t(left)\tand\thyperallometric\t(right)\tZRT scaling\t\ngenes\tin\tcortical-type\tspecific\tcell\tmarkers. 2\tCircle\tsize\tdenotes\tenrichment\tratio,\tsignificant\tassociations\t(p<0.05,\thypergeometric\t test)\tare\t\nhighlighted\twith\tblack\toutline. \t\n\t\nAn\texpanded\tneocortex\tis\ta\thallmark\tof\tthe\tprimate\tbrain.\tA\trecent\ttranscriptomic\tsurvey\tof\tthe\tneocortex\tacross\t\nprimate\tspecies\tidentified\ta\tset\tof\tgenes\tdifferentially\texpressed\tin\thumans\t(hDEGS)\tand\tlocated\tnear\tto\tgenomic\t\nregions\tthat\tare\thighly\tconserved\tacross\tmammals\tbut\tsignificantly\taltered\talong\tthe\thuman\tlineage,\teither\tthrough\t\naccelerated\tDNA\tsubstitution\trates\t(human\taccelerated\tregions;\tHAR)\tor\tdeletions\t(human\tconserved\tdeletions;\t\nhCONDELS).107–109\tWe\ttested\twhether\tthese\tgenes\twere\tassociated\twith\thuman\tneocortical\texpansion\tin\tvivo.\tWe\tfound\t\nthat\tZRTscaling\tgenes\twere\tsignificantly\tenriched\tfor\thDEGs\tlocated\tnear\tHARs\t(overlap\t=\t37;\tenrichment=2.09,\t\nphypergeom<0.0001)\tand\thCONDELS\t(overap=17;\tenrichment=2.0,\tphypergeom=0.008).\tOf\tthese,\t22\t(56%)\twere\texpressed\t\nmore\thighly\tin\tneocortical\tthan\tallocortical\tregions,\tincluding\tseveral\tcell\tadhesion\tmolecules\t(DSCAM,\tPCDH7,\tPCDH9,\t\nLRFN2),\tteneurins\t(TENM3)\tand\tephrins\t(EFNA5),\tas\twell\tas\tgenes\twith\tfunctional\tlinks\tto\tlanguage\tacquisition\t\n(FOXP2)\tand\tneurodevelopmental\tdisorders\t(MEF2C,\tAFF2,\tZEB2)\t(Table\tS9).\t\nProlonged\tneural\tmigration\tprecedes\tfaster\texpansion\tacross\tthe\tneocortex\t\nFocusing\tfurther\ton\tneocortical\texpansion,\twe\tremoved\tallo-\tand\ttransitory\tperiallo-cortical\tstructures\t(hippocampus,\t\nretrosplenial\tcortex,\tentorhinal\tcortex\tand\tpaleocortex)\tand\trepeated\tour\tregional\tcorrelation\tanalysis\tover\tall\tZRT\t\ngenes.\tWithin\tthe\tneocortex,\ta\tsubset\tof\t116\tZRT\tgenes\t(including\t113\tZRTscaling\tgenes)\twere\tsignificantly\tassociated\t\nwith\tdifferential\trates\tof\texpansion\tacross\tneocortical\tregions\t(ZRTneo;\tpFDR<0.01),\twith\tmost\tassociations\tlocalised\tto\t\nthe\tintermediate\tzone\t(IZ;\tFigure\t4a;\tTable\tS10).\tZRTneo\tgenes\twere\talso\tenriched\tfor\thDEGS\tlocated\tnear\tHARs\t\n(overlap=\t10;\tenrichment=2.03,\tphypergeom=0.028)\tincluding\tPCDH7,\tPCDH9,\tTENM3\tand\tAFF2\tbut\tnot\thCONDELS\t(Table\t\nS9).\t\nWe\tobserved\tcontrasting\tcell\ttype\tenrichments\tof\tZRTneo\tgenes\tat\t15\tand\t21\tPCW.\tConsistent\twith\trole\tof\tprolonged\t\nradial\tglial\tproliferation\tin\tproposed\tmodels\tof\tcortical\texpansion,11,51,54\thighly\texpressed\tZRTneo\tgenes\tin\tareas\twith\ta\t\nhigher\trate\tof\texpansion\tover\tgestation\twere\tenriched\tin\tradial\tglia\tand\tintermediate\tprogenitors\tat\t15\tPCW\t\n(phypergeom=0.045,\t0.040\trespectively;\tFigure\tS12;\tTable\tS11)\twith\tsignificant\tassociations\tlocalised\tto\tthe\tcortical\t\nplate,\tsubplate\tand\tsubventricular\tzone\t(Figure\t4a).\tEarly\thyperallometric\tZRTneo\tgenes\tare\tupregulated\tin\tboth\touter\t\n(CDC42EP4,\tHS6ST1)\tand\tventricular\t(FBXO32)\tradial\tglial\tsubpopulations53\t(Figure\tS12).\tIn\tcontrast,\tZRTneo\tgenes\t\nexpressed\tin\tneocortical\tareas\twith\tslower\trelative\tgrowth\twere\tlocalised\tto\tthe\tcortical\tplate\tand\tsubplate\tbut\tnot\t\nspecifically\tenriched\tfor\tany\tmajor\tcell\ttypes\t(all\tphypergeom>0.05;\tTable\tS11).\tHowever,\thypoallometric\tZRTneo\tgenes\t\nwere\texpressed\tby\tneurons\t(NFE2L)\tand\tinvolved\tin\tdendritic\t(ABGRB3110)\tand\tsynaptic\t(NPTX2111)\tplasticity,\t\nindicative\tof\ta\tpopulation\tof\tmaturing,\tnot\tproliferative,\tcells\twith\tneuronal\tlineage\tin\tthese\tregions.\t\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n10 \nAt\t21\tPCW,\tafter\tthe\tpeak\tperiod\tof\tneurogenesis,\twe\tobserved\tthe\topposite\tpattern\tof\tcell\ttype\tenrichments.\tZRTneo\t\ngenes\texpressed\tin\tthe\tIZ\tsubjacent\tto\tpreferentially\texpanded\tcortical\tareas\twere\tenriched\tin\tneuronal\tpopulations\t\n(enrichment\t=\t2.19,\tphypergeom=\t0.00011)\t(Figure\t4a-c)\twhereas,\thypoallometric\tZRTneo\tgenes\twere\tenriched\tin\t\nproliferative\tglial\tcell\ttypes\t(IPC:\tenrichment=2.96,\tphypergeom\t<0.0001;\tRG:\tenrichment=2.36,\tphypergeom<0.0001;\tFigure\t\n4b,c;\tTable\tS11).\tThe\tpresence\tof\tpost-mitotic\tneuronal\tmarkers\tin\tthe\tIZ\tat\t21\tPCW\tsuggested\tthat\tneuronal\t\nmigration\twas\tongoing\tin\tcortical\tareas\twith\tthe\tfastest\trate\tof\texpansion\tin\tthe\tthird\ttrimester\tof\tgestation.\tThis\tis\t\nconsistent\twith\ta\tconserved\tmechanism\tof\tmammalian\tcortical\texpansion\twhereby\tlonger\tneurogenic\tperiods\tlead\tto\t\nan\texpanded\tneocortex.11,112–115\tIn\tthis\tcontext,\ton\tboth\tphylogenetic\tand\tontogenetic\tscales,\tlater\tdeveloping\tcortical\t\nregions\twould\texhibit\tfaster\trates\tof\texpansion.51,115,116\tA\tprominent\thypothesis\tof\tneocortical\texpansion\thas\tsuggested\t\nthat,\tin\tprimates,\tthis\tprocess\tis\trealised\tthrough\tthe\tcontinued\tproduction\tof\tupper\tlayer\tneurons\tfrom\touter\tradial\t\nglia\t(oRG)\tpopulations\tsituated\tin\tthe\touter\tSVZ,\ta\tcell\tpopulation\tgreatly\texpanded\tin\tthe\tprimate\tbrain.51,54\t\t\n\t\n\t\nFigure\t4:\tPreferential\tneocortical\texpansion\tis\tassociated\twith\tdifferential\ttiming\tof\tneurogenesis\tand\tgliogenesis.\ta. \t133\tZRT\tgenes\twere\t\nassociated\t(pFDR<0.01)\twith\tareal\tscaling\tof\tthe\tneocortex\t(after\texcluding\tpaleo -\tand\tarchi-cortex;\tZRTneo).\tMost\tsignificant\tassociations\twere\t\nlocalised\tto\tthe\tIZ.\tb.\tnormalised\t(Z-score)\texpression\tprofiles\tfor\tgenes\tcorrelated\twith\tareal\tscaling\tin\teach\ttissue\tzone\tat\t21\tPCW.\tAssociations\tat\t\n15\tPCW\tare\tshown\tin\tFigure\tS12.\tNegative\tassociations\t(higher\trelative\texpre ssion\tin\thypoallometric\tregions)\tshown\tin\tblue,\tpositive\tassociations\t\nare\tin\tred.\tLighter\tcolours\tindicate\thigher\trelative\texpression.\tMost\tsignificant\tassociations\tare\tin\tthe\tIZ.\t c.\tMid-gestation\tcell\tclusters2\tsignificantly\t\nenriched\t(p<0.01)\tfor\tgenes\tassociated\twith\tareal\tscaling\tin\tthe\tIZ\tat\t21\tPCW.\tTerritories\tof\tthree\tcell\ttypes\tare\tshown.\tNeg ative\tand\tpositive\tZRTneo\t\ngenes\tare\tenriched\tin\tprogenitor\tcells\tand\tneurons,\trespectively\t d.\twedge\tplots\tare\tshown\tfor\ttwo\tZRT neo\tgenes\texpressed\tby\tspecific\tce ll\ttypes:\tMDK\t\n(glial)\tand\tCUX1\t(upper\tlayer\tneurons).\tRows\tindicate\ttissue\tzones\tand\tcolumns\tindicate\tcortical\tregions\tordered\taccording\tto\tallometric\tscali ng\tfrom\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n11 \nhyper\tto\thypoallometric.\tColour\tbar\tindicates\tnormalised\texpression\tlevels\t(a.u.).\t e.\texpression\t(Z-score)\tof\tMDK\tand\tCUX1\tin\tall\tregions\tsampled\tin\t\nthe\tIZ,\tordered\tfrom\thypo\t(top)\tto\thyperallometric\t(bottom)\tscaling.\t f.\tIZ\texpression\tof\tCUX1\t(middle)\tand\tMDK\t(right)\tprojected\tonto\tcorresponding\t\nμBrain\tsurface\tatlas\tlabels\tand\tdisplayed\ton\tthe\t36w\tdHCP\ttemplate\tsurface.\tRegions\twhere\texpression\tfor\ta\tgiven\tgene\twas\tnot \tavailable\tare\tshown\t\nin\tgrey.\tFor\tcomparison,\taverage\tallometric\tscaling\tin\teach\tregion\tis \tdisplayed\t(left).\t\n\t\nTo\texamine\tthis\tproposed\tmechanism\tin\thumans,\twe\tfocused\ton\tCUX1,\ta\tmarker\tof\tlayer\tIII/IV\tneurons\tthat\tregulates\t\ndendritic\tmorphology117\tand\tis\texpressed\thighly\tin\tpreferentially-expanded\tcortical\tregions\t(Figure\t4d-f).\tCUX1\tis\t\nlocated\tdownstream\tof\tHAR426\tand\tpathogenic\tmutations\tin\tCUX1\tare\tassociated\twith\tASD,\tintellectual\tdisability\tand\t\nepilepsy.118,119\tWe\tfind\tthat,\tin\tthe\tIZ\tat\t21\tPCW,\tCUX1\texhibits\texpression\tthat\tvaries\talong\ta\thypo-to-hyperallometric\t\ngradient\t(Figure\t4d,e;\t𝜏=0.52,\tpFDR=0.002).\tTo\tvalidate\tthese\tobservations,\twe\texamine\tISH\tstaining\tof\ta\tsecond\tupper\t\nlayer\tmarker,\tSATB2,\tin\tfive\tregions\twith\tdifferential\tallometric\tscaling,\tfinding\texamples\tof\tupper\tlayer\tSATB2+\t\nneurons\twithin\tthe\tIZ\tof\tregions\twith\ta\tfaster\trate\tof\texpansion\tin\tmid-\tto\tlate-gestation\t(Figure\tS13).\tThe\tprolonged\t\nmigration\tof\tthese\tcell\tpopulations\tin\texpanding\tneocortical\tregions\tis\ta\tpotential\tconsequence\tof\tdifferential\t\nneurogenic\ttiming\tacross\tthe\tneocortical\tsheet\tthat,\tat\tleast\tin\tpart,\tsupports\tthe\taccelerated\texpansion\tof\t\nhyperallometric\tcortical\tregions\tduring\tgestation.\t\nSeveral\tmechanisms\texist\tto\tregulate\tgene\ttranscription\tduring\tearly\tbrain\tdevelopment.120,121\tTo\tidentify\tpotential\t\nregulators\tof\tZRT\tgene\texpression\tin\tthe\tdeveloping\tfetal\tcortex,\twe\tused\ta\trecent\tchromatin\taccessibility\tatlas122\tto\t\nexamine\tthe\tposition\tof\topen\tchromatin\tregions\t(OCR)\tin\tthe\tmid-gestation\tbrain\trelative\tto\tZRT\tgenes.\tWe\tfound\tthat\t\nZRT\tgenes\twere\tmore\tlikely\tthan\tnon-ZRT\tgenes\tto\tbe\tlocated\tnear\tto\tpredicted\tregulatory\telements\t(pREs),\ta\tsubset\tof\t\nOCRs\tthat\tare\tlikely\tto\tfunction\tas\tneurodevelopmental\tenhancers\tin\tmid-gestation122\t(OR:\t1.38\tp\t<\t0.0001;\tFigure\t\nS14;\tTable\tS12).\tMoreover,\tZRTscaling\tand\tZRTneo\tgenes\twere\tsignificantly\tenriched\tfor\tgenes\tlocated\tnear\tto\tpREs\t\n(enrichment\t=\t1.25,\t1.31\tphypergeom<0.0001,\t<0.005\trespectively;\tTable\tS12).\tFocusing\ton\tlaminar\tspecificity\tof\tZRT\t\ngene\texpression,\twe\tfound\tthat\tover\t25%\tof\tZRTneo\tgenes\twere\tlocated\timmediately\tup-\tor\tdownstream\tof\tOCRs\t\nspecific\tto\tthe\tupper\tlayers\tof\tthe\tcortical\tplate,\tcompared\tto\t9%\tlocated\tnear\tto\tdeep\tlayer\tOCRs\t(Figure\tS14B).\t\nTranscription\tfactor\tmotifs\tcontained\twithin\tOCRs\tspecific\tto\tupper\tcortical\tlayers\tand\tproximal\tto\tZRTneo\t(n=20)\t\nincluded\tbHLH,\tLIM\tand\tPOU\thomeobox\tand\tHMG-box\tmotif\tfamilies\t(Figure\tS14C)\tthat\tbind\tto\ttranscription\tfactors\t\nwhich\tregulate\tsuperficial\tneuronal\tidentify\t(e.g.:\tE2A,\tBRN1,\tLHX2).123–125\tThus,\tthe\tdifferential\taccessibility\tof\tspecific\t\nregulatory\telements\tcan\tresolve\tthe\tlaminar\tidentity\tof\tmaturing\tupper-layer\texcitatory\tneurons\tmigrating\tthrough\tthe\t\nIZ\tat\t21\tPCW.\t\t\nBased\ton\tthis\tevidence,\twe\treasoned\tthat\tneuronal\tmigration,\tand\tthus\tneural\tproliferation,\twould\tbe\tcomplete\tor\tnear\t\ncomplete\tat\t21\tPCW\tin\tneocortical\tareas\twith\tslower\texpansion\trates\tin\tthe\tthird\ttrimester.\tIn\tthis\tcase,\texpression\tof\t\nproliferative\tcell\tmarkers\t(Figure\t4)\twould\treflect\tgliogenesis\trather\tthan\tneurogenesis.\tTo\ttest,\twe\tcompared\tZRTneo\t\ngenes\tassociated\twith\tcortical\tscaling\tat\t21\tPCW\tin\tthe\tIZ\tto\tregion-specific\tcell\ttype\tsignatures\tin\tthe\tmid-fetal\tbrain.2\t\nReflecting\tthe\tproximity\tto\tmedial\tallocortex\tand\tperiallocortical\tregions,\twe\tidentified\tseveral\tmidline\tidentity\tgenes\t\n(MID1,\tDMRT5,\tEMX2)\twith\thigh\texpression\tin\thypoallometric\tcortex\tas\twell\tas\tmarkers\tof\tcell\tproliferation\t(HMMR,\t\nHAUS6,\tCENPN,\tCENPH,\tPIK3C3)\t(Figure\t4b;\tTable\tS10).\tIn\tsupport\tof\tour\thypothesis,,\twe\tfound\tthat\thypoallometric\t\nZRTneo\tgenes\twere\tspecifically\tenriched\tin\t(peri)allocortical\tglial\tcell\tpopulations\t(MDK,\tSAP30,\tTMEM98;\tastroglia,\t\nphypergeom=0.01;\tOPC,\tphypergeom=0.07).\t\nTMEM98\tis\ta\tMYRF-interacting\tprotein\tspecifically\texpressed\tin\tnewly-differentiated\toligodendrocytes\tin\tthe\t\ndeveloping\tcentral\tnervous\tsystem126\twhereas\tMDK\tis\ta\tgrowth\tfactor\texpressed\tin\tpre-OPCs\tthat\tcan\tinduce\t\ndifferentiation\tin\toligodendrocyte-lineage\tOL1+\tcells\tin\tvitro.127–129\tSAP30\tforms\ta\tco-repressor\tcomplex\twith\tHDAC1\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n12 \nand\tHDAC2,\tclass\tI\thistone\tdeacetylases\tthat\tregulate\tgene\ttranscription\tand\tare\tessential\tfor\toligodendrocyte\t\nmaturation.130–133\tSimilar\tnegative\tcorrelations\twith\tcortical\texpansion\twere\trecorded\tin\tOPC\tcell\tpopulation\tmarkers\t\nS100B\t(Table\tS10;\t𝜏=-0.33,\tpFDR=0.070),\tNKX2-2\t(𝜏=-0.41,\tpFDR=0.019)\tand\tthe\tglial\tprogenitor\tmarker\tEGFR\t(Figure\t\nS15),\twhich\thas\tbeen\tvalidated\tpreviously\tas\ta\tcritical\tgene\trelated\tto\tbrain\tsize.134\t\tIn\tan\tindependent\tdataset,33\twe\t\nobserved\tsimilar\ttrends\tin\tOLIG1\texpression\tin\tmid-gestation\tacross\tcortical\tregions\twith\tdifferential\tdevelopmental\t\nexpansion\t(Figure\tS16).\tOverall,\tthese\tdata\tsuggest\tthat\tthe\tdevelopmental\ttiming\tof\tthe\tneuro-\tto\tgliogenic\tswitch\t\nvaries\tacross\tthe\tneocortical\tsheet,\twith\tthe\tlength\tof\tthe\tneurogenic\tperiod\tsupporting\tdifferential\trates\tof\tneocortical\t\nexpansion\tduring\tthe\tthird\ttrimester\tof\tgestation.\t\t\nNeocortical\tscaling\tgenes\tare\tcritical\tfor\ttypical\tneurodevelopment\t\nGiven\ttheir\tlikely\timportance\tin\tshaping\tearly\tnormative\tneurodevelopment,\twe\thypothesized\tthat\tthe\tZRTneo\tgenes\t\nwould\tbe\tsusceptible\tto\tseverely\tdisruptive\tmutations\t(i.e.,\tloss-of-function\tvariants).\tWe\tfound\tsignificant\tenrichment\t\nof\thyperallometric\t(median\tloss\tof\tfunction\tobserved/expected\tupper\tbound\tfraction\t(LOEUF)\tscore\t=\t0.26,\t\npermutation\tp\t=\t0.0003\tusing\trandom\tgene\tsets\tof\tsimilar\tsize:\tppermutation)\tbut\tnot\thypoallometric\t(median\tLOEUF\tscore\t\n=\t0.40,\tppermutation\t=\t1)\tZRTneo\tgenes,\tsuggesting\ta\tdisproportionate\tlevel\tof\tvulnerability\tto\tloss-of-function\tvariation\tin\t\ngenes\twhose\texpression\tis\tgreater\tin\tareas\tthat\texpand\tfastest\tin\tthe\tthird\ttrimester.\tWithin\tthese,\twe\tidentified\ta\tset\tof\t\nconstrained\tgenes\texpressed\thighly\tin\tthe\tsubventricular\tzone\tat\t15\tPCW\tin\thyperallometric\tregions.\tThese\tgenes\tare\t\ninvolved\tin\textracellular\tmatrix\tformation\tand\tinteraction\t(EFEMP2,\tLOEUF=0.56,\tPTPRM,\tLOUEF=0.33),\tand\tepithelial-\nto-mesenchymal\ttransition\t(FBXO32,135\tLOEUF=0.64),\tpathways\tcrucial\tto\touter\tradial\tglia\tspecification\tand\t\ndifferentiation\t\tin\tgerminal\tzones\tof\tthe\tdeveloping\tbrain.53\tFollow-up\tanalyses\tusing\tgenome-wide\tmetrics\tfor\tdosage\t\nsensitivity136\tconfirmed\tthe\tenrichment\tof\thyperallometric\tZRTneo\tgenes\tas\thaploinsufficient\t(62%\tof\tgenes,\tppermutation\t<\t\n0.0001\tusing\trandom\tgene\tsets\tof\tsimilar\tsize)\tand\tnot\ttriplosensitive\t(19%,\tppermutation\t=\t0.9418)\t–\ta\thighly\tpathogenic\t\nmechanism\tfor\tloss-of-function\tmutations.\t\nTo\tassess\tthe\tclinical\trelevance\tof\tthese\tdistinct\tZRTneo\tgene\tsets\t(i.e.,\thypoallometric\tand\thyperallometric),\twe\t\nperformed\tenrichment\tanalyses\tusing\tMAGMA137\tacross\tan\tarray\tof\tpreviously\tpublished\tgenome-wide\tassociation\t\nstudies\t(GWAS).\tWe\tfound\tthat\tZRTneo\tgene\tsets\twere\tnot\tenriched\tfor\tbirth\toutcomes\t(gestational\tduration)\tor\t\ncognition\t(educational\tattainment),\tbut\thypoallometric\tZRTneo\tgenes\twere\tenriched\tfor\texternalizing\tbehavior\t(β=0.17\t,\t\np=\t0.007)\tand\thyperallometric\tZRTneo\tgenes\twere\tenriched\tfor\tschizophrenia\t(SCZ;\tβ=0.17,\tp=0.004).\tFurther\tanalysis\t\nusing\tpostmortem\tgene\texpression\tdata\tfrom\tpatients\twith\tneurodevelopmental\tdisorders\trevealed\tsignificant\t\nenrichment\tof\tZRTneo\tgene\tsets\twithin\tmultiple\tco-expression\tmodules.138\tBoth\thypoallometric\tand\thyperallometric\t\nZRTneo\tgenes\twere\tenriched\tin\tcross-disorder\tmodule\tCD1\t(both\tppermutation\t<\t0.05)\t–\tdownregulated\tin\tautism\tspectrum\t\ndisorder\t(ASD),\tSCZ,\tand\tbipolar\tdisorder,\tand\tcontaining\tneuron-enriched\tgenes\tand\tgenes\twith\tASD-\tand\tSCZ-\nassociated\tnonsynonymous\tde\tnovo\tvariants\tfrom\twhole-exome\tsequencing;\tand\thyperallometric\tZRTneo\tgenes\twere\t\nenriched\tin\tmodule\tCD13\t(ppermutation\t<\t0.05)\t–\talso\tdownregulated\tin\tASD,\tSCZ,\tand\tbipolar\tdisorder,\tand\tcontaining\t\nneuron-enriched\tgenes.\t\t\n\t\nDiscussion\t\nDespite\tthe\taltriciality\tof\tthe\thuman\tbrain\tat\tbirth,\tareal\texpansion\tof\tthe\tcortex\tduring\tthe\tsecond\tand\tthird\ttrimester\t\nof\tgestation\tis\tcritical\tfor\tlater\tneurodevelopmental\tfunction.\tCortical\tsurface\tarea\tincreases\texponentially\tduring\tthe\t\nthird\ttrimester\tof\tgestation,\tpermitted\tby\trapid\tcortical\tfolding\tover\tthe\tsame\tperiod.\tPowered\tby\ta\tnew\t3D\tatlas\tof\tthe\t\ndeveloping\tbrain,\tour\tresults\tprovide\ta\tmultiscale\tunderstanding\tof\tfetal\tcortical\texpansion\tin\tthe\tsecond\thalf\tof\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n13 \npregnancy.\tWe\tfind\tthat\tdifferential\texpansion\tof\tcortical\tareas\tin\tgestation\trespects\tanatomical\tand\tevolutionary\t\nboundaries\tbetween\tcortical\ttypes5,9\tand\tis\tsupported\tby\tan\textended\tperiod\tof\tneural\tmigration\tthrough\tmid-\ngestation.51,52,54\t\t\nNeurogenesis\texhibits\ta\tconserved\torder\tbut\tnonlinear\tscaling\tacross\tspecies.139\tLonger\tneurogenic\tperiods\tin\tlarger-\nbrained\tspecies,\tsupported\tby\ta\tlarger\tpool\tof\tprogenitors\tin\tproliferative\tzones,\tresult\tin\tthe\tpreferential\texpansion\tof\t\nlater\tdeveloping\tstructures.11,112–114,139\tIn\tmammals,\tdifferences\tin\tthe\ttiming\tand\trate\tof\tneuron\tproduction\tvary\tacross\t\ncortical\tareas\twith\tevidence\tto\tsuggest\tthat\tprogressive\ttermination\tof\tcortical\tneurogenesis\toccurs\talong\ta\trostral-\ncaudal\taxis.140–142\tIn\tthis\tcase,\tearlier\ttermination\tof\tneuronal\tproduction\tin\tthe\tanterior\tcortex\tcould\tcreate\ta\tpotential\t\naffordance\tfor\tincreased\tneuronal\tsize\tand\tarborisation,\tleading\tto\tincreased\tareal\texpansion\tduring\t\ndevelopment.25,139,143\tHowever,\tfurther\tevaluation\tof\tareal\tdifferences\tin\tneurogenic\ttiming\tin\tthe\tprimate\tcortex\t\npresents\ta\tmore\tcomplex\tpicture,\twith\tneurogenesis\tterminating\tfirst\tin\tlimbic\tand\tallocortical\tstructures\tbut\t\ncontinuing\tin\tthe\tprefrontal\tcortex\tbeyond\tmid-gestation.139,144,145\tCoupled\twith\tthe\tnonlinear\tprogression\tof\thuman\t\ngyrification\tover\tgestation,146\tthis\tsuggests\tthat\tareal\tdifferences\tin\tcortical\tscaling\tare\tlikely\tfounded\tupon\tan\t\nalternative\tschema.49,51\t\nAlternative\thypotheses\thave\tbeen\tput\tforward\ton\tthe\trole\tof\toRG\tproliferation,\tand\tthe\tprolonged\tproduction\tof\t\nneurons\tor\tglia,\tin\tcortical\texpansion.49–52\tOur\tfindings\tdemonstrate\tthat\tprior\tto\tgyrification,\tbut\tafter\tthe\tpeak\tperiod\t\nof\tneurogenesis,\tsupragranular\tneurons\tcontinue\tto\tmigrate\tto\tneocortical\tareas\twith\tthe\tfastest\trate\tof\texpansion\tin\t\nthe\tthird\ttrimester.\tIn\tthe\tprimate\tbrain,\toRGs\tproduce\tlarge\tnumbers\tof\tupper\tlayer\tneurons,\tprovide\ta\tscaffold\tfor\t\nneural\tmigration\tand,\tupon\tcompletion\tof\tneurogenesis,\tact\tas\ta\tsource\tof\tglial\tcells\tin\tmid-\tto\tlate-gestation.49,52,54,147\t\nThus,\tregulation\tof\tneuro-to-gliogenic\ttiming\tin\tthe\toRG\tsubpopulation\tmay\trepresent\ta\tplausible\tcandidate\tfor\t\ndifferential\trates\tof\tneocortical\texpansion49,147\tThough\tpresent\tin\tother\tmammals,\tthe\toSVZ\tis\texpanded\tin\tprimate\t\nspecies51,53,54\tand\tproliferation\tin\tthe\toSVZ,\tmarked\tby\tmitotic\tactivity,\tis\thighest\tin\tregions\tthat\texpand\tmost\tin\tlater\t\ndevelopment.148\tWhile\tour\tdata\tsuggest\trapid\tareal\texpansion\tis\tpreceded\tby\tan\textended\tneurogenic\tperiod,\twe\tlack\t\nthe\tdata\tto\tconfirm\ta\tsimilarly\textended\tperiod\tof\t\tgliogenesis.\tIn\thumans,\tneurogenesis\tprecedes\tcortical\tfolding\twith\t\nthe\tsubsequent\tgliogenic\tperiod\tmore\tclosely\taligned\tto\tthe\ttiming\tof\tcortical\texpansion.49,50\tAn\textended\tneurogenic\t\nperiod\tcoupled\twith\ta\tlonger\tmigration\ttime\tdue\tto\tthe\texpanding\tvolume\tof\tthe\tbrain\tmay\tnecessitate\tan\textended\t\ngliogenic\tperiod\tto\tpopulate\tthe\texpanding\tneuropil.50\tEvaluating\tthe\ttemporal\tand\tspatial\tregulation\tof\tglial\tfate\t\ntransition\tand\tproliferation\tin\tthe\toSVZ\tduring\tthe\tsecond\thalf\tof\tgestation\trepresents\ta\tcritical\tnext\tstep\tin\t\nunderstanding\tthis\tprocess.\t\nIn\tgyrencephalic\tspecies,\tthe\tbuckling\tand\tfolding\tof\tthe\tcerebral\tcortex\tallows\tfor\tincreased\tsurface\tarea\tof\tthe\tcortical\t\ngrey\tmatter.\tGreater\ttangential\texpansion\tof\tsuperficial\tcortical\tlayers\trelative\tto\tsubcortical\ttissue\trepresents\ta\tcore\t\nfeature\tof\tbiomechanical\tmodels\tof\tcortical\tgrowth\tand\tfolding.149–152\tHowever,\tuniform\trates\tof\ttangential\texpansion\t\ncan\tnot\tfully\taccount\tfor\tthe\tconsistency\tin\tlocation\tof\tcortical\tfolds\tacross\tindividuals,\twith\tadditional\tgenetic\t\ncontributions\tto\tgyral\tpatterning\tclearly\tdemonstrated\tin\ttwin\tstudies.153,154\tIn\tcontrast,\tgenetically\tdetermined\tareal\t\ndifferences\tin\texpansion\trate\tmay\tgive\trise\tto\tthe\tconsistent\tpatterns\tof\tfolding\tobserved\tacross\tthe\tneocortical\t\nsheet.148,155\tRecently,\tlarge-scale\tneuroimaging\tstudies\thave\tidentified\tpatterns\tof\taltered\tcortical\tmorphometry\tthat\t\nare\tshared\tacross\tcommon\tneuropsychiatric\tconditions\tand\thuman\tgenetics\tstudies\thave\tbegun\tto\tconverge\ton\t\nputative\tmechanisms\tunderlying\tcortical\tabnormalities\tin\tdevelopmental\tgenetic\tdisorders.156–160\t\tHere,\twe\tidentify\t\nsignificant\tenrichment\tof\tpathogenic\tloss-of-function\tvariants\tin\tgenes\tthat\tare\texpressed\tin\tmid-gestation,\tlinked\tto\t\nspecification\tof\touter\tradial\tglia\tand\tassociated\twith\tdifferential\trates\tof\tcortical\texpansion.\tTaken\ttogether,\tthese\t\nfindings\tsuggest\tthat\tthere\tare\ttemporal\twindows\tof\tsusceptibility\tin\tthe\tearly\tstages\tof\tbrain\tdevelopment\twhere\tareal\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n14 \ndifferences\tin\tthe\ttiming\tof\tfundamental\tneurogenic\tprocesses\tcould\tunderlie\tobservable\tcortical\tabnormalities\tand\t\npostnatal\tfunctional\tpathologies\tin\tneurogenetic\tdisorders.47\t\nSpatially-embedded\tgene\texpression\tatlases\tof\tthe\tadult\thuman35,68\tand\tmouse70,161\thave\tproven\texceptionally\tpowerful\t\nin\trecent\tyears,\tbridging\tresolution\tgaps\tto\tcommon\tneuroimaging\tmodalities162,163\tand\tproviding\tinsight\tinto\tthe\t\nmolecular\tcorrelates\tof\tstructural26,42,164\tand\tfunctional\tneuroanatomy,36,165,166\tbrain\tdevelopment,167–169\tdisease\tand\t\ndisorder.47,170,171\tIn\tsuch\tstudies,\tcomparisons\twith\tin\tvivo\tneuroanatomy\tcan\tonly\tbe\tfully\trealised\tthrough\tthree-\ndimensional\tlocalisation\tof\ttissue\tsamples\twithin\ta\tcommon\tcoordinate\tspace.62,68,70\tTo\tdate,\ta\tlimitation\tof\tthis\t\napproach\thas\tbeen\teither\tthe\tsampling\tof\ta\tnarrow\tage\trange\toutside\tof\tkey\tdevelopmental\tperiods35,69\tor,\tin\t\ndevelopmental\tdatasets,\ta\tlack\tof\t3D\tspatial\tinformation3\tand\trelatively\tcoarse\tanatomical\tsampling.34\tTo\tfill\tthis\tgap,\t\nwe\tprovide\ta\tnew\tresource,\tμBrain,\tbuilt\tupon\texisting\topen-source\tdata,\tto\tallow\tresearchers\tto\tmap\tdevelopmental\t\nneuroanatomy\tof\tthe\thuman\tfetal\tbrain\tonto\tearly\thistogenic\tprocesses\tusing\tcontemporaneous\tpost\tmortem\tdata.\tThe\t\nreconstructed\t3D\tμBrain\tatlas\tbrings\tdetailed\ttissue\tmicroarray\tand\tin\tsitu\thybridisation\tdata\tinto\talignment\twith\ta\t\ndevelopmental\tanatomical\tatlas\tof\tthe\tfetal\tbrain.89\tThe\tμBrain\tatlas\twill\tenable\tfuture\tstudies\tto\texamine\ttissue\tor\t\nregion-specific\texpression\tsignatures\tin\trelation\tto\taspects\tof\tstructural\tor\tfunctional\tbrain\tdevelopment\tin\tutero\tor\t\nidentify\tspatial\tor\ttemporal\twindows\tof\tvulnerability\tfor\tgenetic\tor\tneurodevelopmental\tdisorders.\t\t\nDevelopmental\tMRI\tstudies\tprovide\tunique\tinsight\tinto\tearly\thuman\tbrain\tdevelopment.\tDue\tto\tlarge\tdifferences\tin\t\nsize,\tshape\tand\ttissue\tcontrast,\tspecialised\ttools\tare\trequired\tfor\tthe\tanalysis\tof\tinfant\tand\tneonatal\tMRI.\tSimilarly,\twe\t\ncannot\trely\ton\tcommon\tcortical\tatlases\tthat\tare\tbased\ton\tadult\tneuroanatomy.87,172\tHere,\twe\tused\tannotations\tderived\t\nfrom\tcytoarchitecture\tof\tthe\tmid-fetal\tbrain\tto\tgenerate\ta\tnew\tcortical\tatlas\tto\tfacilitate\tfurther\tresearch\tin\tearly\tbrain\t\ndevelopment.\tA\tkey\tarea\tfor\tfuture\tresearch\tin\tthis\tfield\tis\tthe\tdevelopment\tand\tvalidation\tof\timproved\tmethods\tto\t\nalign\tearly\tMRI\tto\tcommon\ttemplate\tspaces.\tThe\tgeometry\tof\tthe\tfetal\tcortex\tis\tsmooth,\tmaking\talignment\tof\tcortical\t\nmorphometry\tan\till-posed\tproblem.\tNewer,\tanatomically-constrained\tregistration\ttechniques\tand\tlarger\tlongitudinal\t\ncohorts\twith\tmultiple\tscans\tduring\tmid-\tto\tlate-trimester\twill\tenable\tmore\tprecise\testimates\tof\tcortical\texpansion\tin\t\nthe\tfuture.90,95\t\t\nWith\tincreasingly\tgranular\tsurveys\tof\tthe\tdeveloping\tbrain\tat\ta\tsingle-cell\tlevel2,107\tthe\tadvent\tof\tspatial\ttranscriptomic\t\ntechnologies,173\tand\ta\tseries\tof\tlarge-scale\tand\topen-access\tperinatal\tneuroimaging\tstudies,84,174,175\twe\tanticipate\t\nμBrain\twill\tprovide\ta\tfoundation\tfor\tdevelopmental\tand\tcomparative\tneuroscience\tto\tintegrate\tand\ttransfer\tknowledge\t\nof\tearly\tbrain\tdevelopment\tacross\tdomains,\tmodel\tsystems\tand\tresolution\tscales.\t\t\n\t\nAcknowledgements\t\nThis\tresearch\twas\tsupported\tby\tthe\tNational\tHealth\tand\tMedical\tResearch\tCouncil\t(NHMRC)\t[1194497\tto\tG.B.],\tthe\t\nMurdoch\tChildren’s\tResearch\tInstitute,\tthe\tRoyal\tChildren’s\tHospital,\tDepartment\tof\tPaediatrics,\tThe\tUniversity\tof\t\nMelbourne\tand\tthe\tVictorian\tGovernment's\tOperational\tInfrastructure\tSupport\tProgram.\tThe\tproject\twas\tgenerously\t\nsupported\tby\tRCH1000,\ta\tunique\tarm\tof\tThe\tRoyal\tChildren’s\tHospital\tFoundation\tdevoted\tto\traising\tfunds\tfor\t\nresearch\tat\tThe\tRoyal\tChildren’s\tHospital.\tL.\tZ.\tJ.\tW\twas\tsupported\tby\tthe\tCommonwealth\tScholarship\tCommission,\t\nUnited\tKingdom.\tV.\tKa.\twas\tsupported\tby\tan\tMRC\t(UK)\taward\tMR/V036874/1\tand\tThe\tDeveloping\tHuman\tConnectome\t\nProject.\tE.C.R.\twas\tsupported\tby\ta\tWellcome\tCollaborative\tAward\t(215573/Z/19/Z).\tAA-B\tand\tJS\twere\tsupported\tby\t\nR01MH132934.\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n15 \nNeuroimaging\tdata\twere\tprovided\tby\tthe\tDeveloping\tHuman\tConnectome\tProject,\tKCL-Imperial-Oxford\tConsortium\t\nfunded\tby\tthe\tEuropean\tResearch\tCouncil\tunder\tthe\tEuropean\tUnion\tSeventh\tFramework\tProgramme\t(FP/2007-2013)\t\n/\tERC\tGrant\tAgreement\tno.\t[319456].\tWe\tare\tgrateful\tto\tthe\tfamilies\twho\tgenerously\tsupported\tthis\ttrial.\t\t\nWe\tare\tgrateful\tto\tthe\tAllen\tInstitute\tand\tassociated\tInvestigators\tfor\tthe\tprovision\tof\tthe\tAtlas\tof\tthe\tDeveloping\t\nHuman\tBrain.\t\t\nAuthor\tcontributions\t\nG.B.,\tS.O.\tV.K.,\tL.Z.J.W.,\tV.K.,\tA.P.,\tJ.V.H.,\tJ.H.,\tE.C.R.\t&\tJ.S.\tperformed\tdata\tacquisition\tand\tdata\tprocessing.\tG.B.,\tS.O.\t&\tJ.S\t\nperformed\tdata\tanalysis.\tG.B,\tJ.S.,\tV.K.,\tL.Z.J.W\t&\tE.C.R.\tcontributed\tto\tmethodology.\tG.B.,\tM.L.S.,\tA.A-B.,\tJ.V.H.,\tA.D.E.,\t\nE.C.R.\t&\tJ.S.\tprovided\tresources\tand\tsupervision.\tE.C.R.\tprovided\tsoftware.\tProject\tconceptualisation:\tG.B\t&\tJ.S.\tWriting\t\ndrafts,\trevisions\tand\tediting:\tall\tauthors.\t\t\t\nConflicts\tof\tinterest\t\nJS\tand\tAFA-B\tare\tco-founders\tof\tCentile\tBioscience.\t\n\t\nMaterial\tand\tMethods\t\nPublic\tdata\tsources\t\nSource\tdata\tunderlying\tthe\tμBrain\tatlas\twere\tmade\tavailable\tas\tpart\tof\tthe\tBrainSpan\tDeveloping\tBrain\tAtlas\t\n[https://atlas.brain-map.org/atlas?atlas=3]\twith\tdetailed\ttissue\tprocessing\tprotocols\tavailable\tfrom\tDing\tet\tal.3\tIn\tbrief,\t\na\tsingle\tprenatal\tbrain\tspecimen\t(21\tPCW;\tfemale)\twas\tbisected\tand\tthe\tright\themisphere\tused\tfor\tserial\tsectioning.\t\nThe\tbrain\tspecimen\twas\tcut\tinto\tfour\tcoronal\tslabs\tand\tfrozen\tin\tisopentane.\tSerial\tcoronal\tsectioning\tat\t20μm\t\nthickness\twas\tperformed\tslab-by-slab\twith\tsequential\tsections\tsubmitted\tto\tNissl,\tAChE\tor\tISH\tstaining\twith\t43\tgene\t\nprobes\tand\tstained\tsections\tdigitally\tscanned\tat\t1μm\t/\tpixel\tresolution.\tIn\ttotal,\t81\tout\tof\t174\tNissl-stained\tsections\t\nwith\tvarying\tsampling\tdensities\t(~0.5mm\tto\t1.2mm\tbetween\tsections)\twere\tselected\tfor\tannotation.3\tExpert\t\nanatomical\tannotations\twere\tconducted\tmanually\ton\teach\tsection.\t\tNissl-\tand\tISH-stained\tsections\twith\tcorresponding\t\nanatomical\tlabels\twere\tmade\tavailable\tfor\tdownload.\tAnatomical\tannotations\twere\talso\tused\tto\tguide\tlaser\t\nmicrodissections\tfor\tDNA\tmicroarray\tanalysis\tacross\tthe\tdeveloping\tcerebral\ttissue\tin\tthe\tleft\themisphere\tof\t4\tseparate\t\nmid-gestation\tspecimens\t(see\tMicroarray\tData\tbelow).\tThe\tsection\tnumbers\tand\tapproximate\tcoronal\tpositions\tof\t\nsections\tused\tin\tthe\tconstruction\tof\tthe\t3D\tμBrain\tatlas\tare\tlisted\tin\tTable\tS1.\t\nImage\tprocessing\t\nWe\tdownloaded\teach\thigh-resolution\tNissl-stained\tsection\t(n=81;\tdownsampled\tto\t2μm/pixel)\tas\tRGB\timages\tin\tJPG\t\nformat\twith\tcorresponding\tanatomical\tlabels\tas\tSVG\tfiles.\t\t\nAfter\tconverting\tSVG\tto\tRGB\tPNG\tformat,\twe\tmanually\tcombined\tanatomical\tlabels\taccording\tto\tthe\thierarchical\t\nontology\tof\tthe\treference\tatlas3\tto\tcreate\ttwo\tcompact\tannotations,\tone\tfor\timage\trepair\tcomprising\t20\ttissue\tstructure\t\nlabels\t(brain-labels)\tand\tone\tfor\tstatistical\tanalysis\tcontaining\tonly\tcortical\tlabels\t(cortex-labels,\tn=30,\tincluding\tone\t\ngeneric\t‘brain\ttissue’\tlabel\tfor\tnon-cortical\tstructures;\tsee\tTable\tS2).\tDue\tto\tthe\tsmall\tsize\tand\tdegree\tof\tmissing\tdata\t\nprecluding\treconstruction,\tmarginal\tzone\tand\tsubpial\tgranular\tzones\twere\tnot\tconsidered\tin\tthis\tanalysis.\tNissl-stained\t\nsections\tand\tcorresponding\tlabel\timages\twere\tthen\tdownsampled\tto\t20μm\t/pixel\tresolution.\t\nHistological\treconstruction\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n16 \nPix2pix\tis\ta\tconditional\tgenerative\tadversarial\tnetwork\t(GAN)\ttrained\tto\tperform\timage-to-image\ttranslation\tbetween\t\npairs\tof\timage\texamples.59\t\tWe\tused\tthe\tpix2pix\tarchitecture\t(Figure\t1b)\tto\tsynthesise\tNissl-stained\timages\tfrom\tlabel\t\nannotations\tin\torder\tto\treplace\tartefacts\twithin\ttissue\tsections\t(Figure\t1c,d).\tFollowing\tconventional\tGAN\tstructure,\t\t\nthe\tmodel\tcombines\ta\tgenerator\tnetwork,\t𝐺,\twith\ta\tclassifier\t(or\tdiscriminator,\t𝐷)\twhose\tobjective\tis\tto\tdetermine\tif\t\nimages\tare\treal\tor\tfake\t(Figure\t1b).\tGAN\ttraining\tis\tperformed\tadversarially\twith\tthe\tgenerator\tnetwork\tcompeting\tto\t\ngenerate\tmore\tand\tmore\trealistic\tsynthetic\timage\tfrom\tlabel\tannotations,\tand\tthe\tdiscriminator\tworking\tto\t\ndiscriminate\tbetween\treal\tand\tfake\texamples.\tAn\t𝐿!\tregularisation\tterm\tis\tadded\tto\tenforce\tthat\tgenerated\timages\tare\t\nas\tclose\tas\tpossible\tto\tthe\tground\ttruth.\tFull\tmodel\tarchitecture\tand\ttraining\tdetails\tare\tincluded\tin\tthe\tSupplemental\t\nMethods.\t\nImage\trepair\t\nTo\tperform\trepair\tof\twhole\tsections,\twe\tsplit\teach\tlabel\timage\tinto\tpatches\tof\t256\t × \t256\tpixels\twith\tan\t8\tpixel\toverlap\t\nand\tpassed\tthem\tthrough\tthe\ttrained\tgenerator.\tThe\tresulting,\tsynthetic\tNissl\tcontrast\tpatches\twere\tstitched\ttogether\t\ninto\ta\tfull\tsection\tmatching\tthe\tdimensions\tof\tthe\toriginal\timage\t(Figure\t1d).\tPatch\tprediction\tand\timage\t\nreconstruction\twas\tperformed\tusing\tMightyMosaic\t[https://pypi.org/project/MightyMosaic/].\t\t\nTo\tdetect\tregions\tof\tthe\toriginal\tNissl-stained\tsection\tthat\tneeded\trepair,\twe\tdesigned\tan\tautomated\toutlier\tdetection\t\nmethod\tbased\ton\tthe\tMedian\tAbsolute\tDeviation\t(MAD)\tof\tpixel\thue\tand\tsaturation.\tThe\toriginal\tNissl-stained\tsections\t\nand\tcorresponding\tGAN-generated\tpredictions\twere\ttransformed\tto\tHSV\tformat\tand\tblurred\twith\ta\tbox\tfilter\t(width\t=\t\nheight\t=\t5\tpixels).\tWe\tidentified\toutliers\twith\tmedian\tabsolute\tdifferences\tin\thue\tand\tsaturation\tbetween\tpixels\tin\tthe\t\nground\ttruth\timage\tand\tits\tsynthetic\tequivalent\tin\thue\tand\tsaturation\tgreater\tthan\tthreshold,\t𝜃,\tset\tto\t2.5,\twhereby\t\nlowering\t𝜃\twould\tincrease\tthe\tnumber\tof\tpixels\tmarked\tas\toutliers.\t\nFor\teach\tsection,\ta\tbinary\tmask\twas\tcreated\tcontaining\tall\tpixels\tidentified\tas\toutliers\tin\tboth\thue\tand\tsaturation.\tA\t\nfinal\topening\toperation\twas\tapplied\tto\tthe\toutlier\tmask\tusing\tan\telliptical\tfilter\t(iterations\t=\t3,\twidth\t=\t3\tpixels)\tto\t\nremove\tspeckles\tin\tthe\tmask.\tIdentified\toutlier\tpixels\twere\tthen\treplaced\twith\tthe\tcorresponding,\tintensity-matched\t\npixels\tfrom\tthe\tsynthetic\timage\tusing\tPoisson\timage\tediting\tto\teffect\timage\trepair\t(Figure\t1d).61\tOutlier\tdetection\tand\t\nrepair\twas\tperformed\tin\tPython\tusing\tOpenCV\t(4.5.2)\t[https://opencv.org/].\t\nμBrain\tvolume\tconstruction\t\nFollowing\tautomated\trepair\tof\tmajor\ttissue\tartefacts\tpresent\tin\tthe\thistological\tdata,\twe\taimed\tto\tdevelop\ta\t3-\ndimensional\treconstruction\tof\tthe\tfetal\tbrain\tto\tfacilitate\tcomparison\twith\tin\tvivo\tMR\timaging\tdata.\tImage\talignment\t\nand\treconstruction\tsteps\tare\tsummarised\tbelow.\tFull\tdetails\tare\tincluded\tin\tSupplemental\tMethods.\t\nSlice-to-slice\talignment\t\nUsing\tthe\tmiddle\tsection\tas\ta\treference,\trepaired\tNissl-stained\tsections\twere\taligned\tusing\ta\tgraph-based,\tslice-to-slice\t\nregistration.176,177\tPairwise\trigid\ttransforms\twere\testimated\tbetween\teach\tsection\tand\tits\tneighbouring\tsections\tin\tthe\t\ndirection\tof\tthe\treference.\tDijkstra’s\tshortest-path\talgorithm\twas\tthen\tused\tto\tcalculate\tthe\tset\tof\ttransforms\twith\t\nlowest\tcost\tto\talign\ta\tgiven\tsection\tto\tthe\treference.176,177\tThe\tselected\ttransforms\twere\tcomposed\tand\tapplied\tto\tboth\t\nthe\timage\tand\tits\tcorresponding\tlabels\tto\tbring\tall\tsections\tinto\tapproximate\talignment\t(Figure\t1e;\tFigure\tS17a).\t\nAffine\tregistration\tto\ta\tfetal\tbrain\tshape\treference\t\nReconstructing\t3D\tvolumes\tfrom\tthe\tconsecutive\talignment\tof\t2D\tsections\tcommonly\tproduces\tan\tartefact\ttermed\t‘z-\nshift’\tcaused\tby\tthe\tpropagation\tof\tregistration\terrors\tbetween\tadjacent\tslices\tand\tresulting\tin\ta\tdistorted\tthree-\ndimensional\tstructure\tin\tthe\tfinal\tvolume.178\tTo\tovercome\tthis\teffect,\tit\tis\tcommon\tto\tuse\ta\tshape\tprior\tto\tguide\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n17 \nregistration\tand\tpreserve\t3D\tshape.62,178,179\tIn\tlieu\tof\ta\tground-truth\tvolume\tfor\tthe\tsectioned\tdata,\twe\temployed\ta\t\npopulation-based\taverage\tanatomical\timage:\tspecifically\tthe\t22-week\ttimepoint\tof\tthe\tGholipour\tet\tal.\tspatio-temporal\t\nfetal\tMRI\tatlas\t(Figure\tS3).63\t\nAfter\tmatching\tMRI-based\ttissue\tlabels\tto\tthe\tμBrain\ttissue\tlabels,\twe\tupsampled\tthe\tMRI\ttemplate\tto\t50μm\tisotropic\t\nresolution\tand\tconverted\tthe\tMRI\tlabels\tinto\tan\timage\tNissl-like\tcontrast\tusing\tthe\ttrained\tGAN\tmodel\t(Figure\tS3c-d).\t\nNissl-contrast\timages\twere\tre-stacked\tinto\ta\t3D\tvolume\tto\tact\tas\tan\tanatomical\tprior\tfor\tregistration.\t\nWe\tperformed\tan\titerative\taffine\tregistration\tprocedure\tbetween\tthe\tMRI-based\tshape\tprior\tand\tthe\t3D\tstack\tof\t\nhistological\tsections.176\tThis\tprocess\twas\trepeated\tfor\ta\ttotal\t5\titerations,\tproducing\ta\tfinal\t3D\tvolume\twith\taligned\t\ncoronal\tslices\tand\ta\tglobal\tshape\tapproximately\tmatched\tto\tthe\tin\tutero\tfetal\tbrain\t(Figure\t1e;\tFigure\tS17a).\t\nFinal\ttemplate\tconstruction\t\nTo\tcreate\tthe\tfinal\t3D\tvolume,\twe\temployed\ta\tdata\taugmentation\ttechnique,\tgenerating\tn=50\tunique\trepresentations\tof\t\nthe\taffinely-aligned\tdata\tby\tapplying\tnonlinear\tdistortions\talong\tall\tthree\timage\taxes.\tFor\teach\tvolume,\twe\tperformed\ta\t\nweighted\tnonlinear\tregistration\tbetween\tneighbouring\tsections\tto\taccount\tfor\tresidual\tmisalignments.\tFinally,\tto\tcreate\t\na\tsmooth\t3D\treconstructed\tvolume,\twe\tco-registered\tall\t50\taugmented\tand\taligned\tvolumes\tinto\ta\tsingle\tprobabilistic\t\nanatomical\ttemplate\twith\tvoxel\tresolution\t150\t×\t150\t×\t150μm\tusing\tan\titerative,\twhole-brain\tnonlinear\tregistration\t\n(Figure\t1e;\tFigure\tS17a;\tSupplemental\tMethods).\tAll\timage\tregistration\twas\tperformed\tin\tPython\t3.7\tusing\tantspyx\t\n(0.2.7).14\t\nCortical\treconstruction\t\nTo\treconstruct\tthe\tfetal\tcortical\tsurface,\twe\tadapted\texisting\tprotocols\tfor\tex\tvivo\t\n[https://freesurfer.net/fswiki/ExVivo]\tand\tnon-human\tprimate\t[https://prime-re.github.io/]\tsurface\treconstruction\t\nwith\tFreesurfer.15\tWe\tused\tthe\tμBrain\ttissue\tlabels\tto\tgenerate\ta\t‘white\tmatter’\tmask\t(all\tsubcortical\tstructures\tand\t\ntissue\tzones,\texcluding\tthe\tcortical\tplate).\tWe\tused\tthis\tmask\tto\tgenerate\tinner\tand\touter\tsurfaces\tfor\tthe\tμBrain\t\nvolume\t(Figure\t1f).\tSurfaces\twere\tsmoothed\tand\tinspected\tfor\ttopological\terrors.\tAll\tprocessing\twas\tperformed\twith\t\nFreesurfer\t(7.3.2).\t\nIn\tsitu\thybridisation\t\nIn\taddition\tto\tserial\tNissl\tstaining,\tinterleaved\tcoronal\tsections\twere\tused\tfor\tin\tsitu\thybridisation\t(ISH)\tof\ta\tseries\tof\t\nneurodevelopmental\tmarker\tgenes\t(Table\tS3).3\tHigh-throughput\tISH\tstaining\twas\tperformed\tfor\teach\tgene,\twith\t\nstained\tsections\tdigitised\tat\t1𝜇m\tresolution.\tQuantification\tof\tthe\tintensity\tof\texpression\tdetection\twas\tperformed\t\nusing\tan\tautomated\tprocedure\tthat\tpseudo-colour\tcoded\tlevels\tof\texpression\tfor\tvisualisation,\twith\tlow-to-high\t\nexpression\trepresented\tas\tblue-to-red.161\t\nCompared\tto\tNissl-stained\tsections\t(n=79\tafter\tquality\tcontrol),\tfewer\tISH\tstained\tsections\twere\tavailable\tfor\teach\tgene\t\n(mean\tn\t=\t41\tafter\tquality\tcontrol),\tprecluding\ta\tfull\t3D\treconstruction\tof\teach.\tWe\tdownloaded\teach\tset\tof\tISH-stained\t\nsections\tand\tremoved\tany\twith\tlarge\tartefacts\t(tearing,\tfolding,\tmissing\ttissue).\tFrom\teach\tfalse-colour\texpression\tmap,\t\nwe\textracted\tthe\tred\tchannel\tto\tfocus\tonly\ton\thigher\texpressing\tcells.\tEach\tsection\twas\tregistered\tto\tthe\tnearest,\t\nrepaired\tNissl-stained\tsection\tusing\taffine\tregistration.\tRegistrations\twere\tvisually\tinspected\tand\tany\tfailures\tremoved.\t\nAligned\tsections\twere\tthen\tstacked\ttogether,\twith\tblank\tslices\tin\tplace\tof\tmissing\tsections\tand\treconstructed\tinto\ta\t3D\t\nvolume\tusing\tthe\tpreviously\tcalculated\tslice-to-volume\talignments\tfor\teach\tsection\t(see\t‘μBrain\tvolume\t\nconstruction’).\t\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n18 \nMicroarray\tdata\t\nWe\tdownloaded\tprenatal\tLMD\tmicroarray\tdata\tfrom\tthe\tBrainSpan\tdatabase\t[https://www.brainspan.org/].\tFor\t\ndetails\ton\ttissue\tprocessing\tand\tdissection\tsee\tMiller\tet\tal.4\tand\tthe\ttechnical\twhite\tpaper\tavailable\tat:\t\n[https://help.brain-map.org/download/attachments/3506181/Prenatal_LMD_Microarray.pdf].\tIn\ttotal,\tnormalised\t\nmicroarray\tdata\tfrom\t58,692\tprobes\tin\t1206\ttissue\tsamples\twere\tavailable\tto\tdownload,\tobtained\tfrom\tthe\tleft\t\nhemisphere\tof\tfour\tpost-mortem\tfetal\tbrain\tspecimens\t(age\t15-21\tPCW,\t3\tfemale).4\tEach\tprobe\twas\tassigned\ta\t‘present’\t\nor\t‘absent’\tannotation\tbased\ton\tstrength\tof\taverage\tprobe\texpression\tover\tcorresponding\tbackground\tsignal.\tThrough\t\ncomparison\twith\tthe\tBrainSpan\treference\tatlas,\twe\tmatched\teach\ttissue\tsample’s\tanatomical\tlabel\tto\ti)\tcorresponding\t\ncortical\tlabels\tincluded\tin\tthe\tμBrain\tatlas\tand\tii)\tone\tof\tfive\ttissue\tzones\t(cortical\tplate,\tsubplate,\tintermediate\tzone,\t\nsubventricular\tzone,\tventricular\tzone)\t(Table\tS4;\tFigure\tS4).\tSamples\tthat\tcould\tnot\tbe\tmatched\tto\tlabeled\tregions\tin\t\nthe\tcortical\tplate\tor\tcorresponding\tsubjacent\ttissue\tzones\twere\tremoved,\tincluding\tsamples\tfrom\tsubcortical\tnuclei,\t\nmidbrain\tstructures\tand\tbrainstem.\t\nMicroarray\tprocessing\t\nWe\tupdated\tgene\tassignments\tfor\tthe\tAllen\tmicroarray\tprobes\tusing\tRe-Annotator180\tand\tremoved\tany\tprobes\t\nassigned\tto\tmore\tthan\tone\tgene,\tresulting\tin\ta\tprobe\tset\t(n=46,156)\tmapped\tto\t20,262\tunique\tgenes.\tLow\tsignal\tprobes\t\ndesignated\t‘absent’\twere\tremoved\t(34.67%\tof\tprobes),\tas\twere\ttissue\tsamples\tfrom\tthe\tmarginal\tzone,\tsubpial\tgranular\t\nzone\tand\tsubcortical\tand\tmidbrain\tstructures\t(54.46%\tof\tsamples).\tWhere\tmultiple\tprobes\tmapped\tto\ta\tsingle\tgene,\t\nthe\tprobe\twith\tthe\thighest\tdifferential\tstability\t(DS),181\tthe\taverage\tpairwise\tcorrelation\tbetween\ttissue\tsample\t\nexpression\tover\tall\tspecimens,\twas\tassigned.\tProbes\twith\tDS<0.2\twere\tremoved.\t\nWhere\tmore\tthan\tone\tsample\twas\tavailable\tfor\ta\tgiven\tregion\tor\tzone,\te.g.:\tsamples\tfrom\tthe\touter\tand\tinner\tcortical\t\nplate\tin\tthe\tsame\tregion,\tgene\texpression\twas\taveraged\tacross\tsamples.\tFinally,\tany\tprobes\twith\tmissing\tdata\tin\tmore\t\nthan\t10%\tof\ttissue\tsamples\twere\tremoved\t(n=1253).\tThis\tresulted\tin\texpression\tdata\tfrom\t8771\tgenes\tacross\t27\t\nregions\tand\t5\ttissue\tzones\tfor\tanalysis\t(Figure\tS4).\t\nFetal\tMRI\t\t\nTo\tmeasure\tcortical\texpansion\tin\tutero\tduring\tthe\tthird\ttrimester,\twe\tanalysed\thigh-resolution\tMRI\tfrom\ta\tlarge\tcohort\t\nof\tfetuses.\t\t\nMRI\tacquisition\t\nFetal\tMRI\tdatasets\t(n=240\tscans\tfrom\t229\tfetuses\taged\tbetween\t21+1\tand\t38+2\tgestational\tweeks+days\t)\twere\tacquired\tas\t\npart\tof\tthe\tDeveloping\tHuman\tConnectome\tProject\t(dHCP)\tusing\ta\tPhilips\tAchieva\t3T\tsystem,\twith\ta\t32-channel\tcardiac\t\ncoil\tin\tmaternal\tsupine\tposition.\tStructural\tT1-weighted\t(T1w),\tT2w,\tfunctional\tMRI\tand\tdiffusion\tMRI\tdata\twere\t\nacquired\tfor\ta\ttotal\tscan\ttime\tof\tapproximately\t45\tminutes.85\tT2-weighted\tSSTSE\tvolumes\twere\tacquired\twith\t\nTE=250ms,\tacquisition\tresolution\t1.1\tx\t1.1mm,\tslice\tthickness\t2.2mm,\t-1.1mm\tgap\tand\t6\tstacks.\tAll\t3D\tbrain\timages\t\nwere\treconstructed\tusing\ta\tfully\tautomated\tslice-to-volume\treconstruction\t(SVR)\tpipeline86\tto\t0.5mm\tresolution\tand\t\nreoriented\tto\tthe\tstandard\tradiological\tspace.\t\nThe\tstudy\twas\tapproved\tby\tthe\tUK\tHealth\tResearch\tAuthority\t(Research\tEthics\tCommittee\treference\t452\tnumber:\t\n14/LO/1169)\tand\twritten\tparental\tconsent\twas\tobtained\tin\tevery\tcase\tfor\timaging\tand\topen\tdata\trelease\tof\tthe\t\nanonymized\tdata.\tAll\tdata\twas\tacquired\tat\tSt\tThomas\tHospital,\tLondon,\tUnited\tKingdom.\t\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n19 \nAfter\timage\tprocessing\tand\tquality\tcontrol,\tthe\tfinal\tdataset\tcomprised\tn=195\tfetal\tMRI\tdatasets\tacquired\tfrom\tn=190\t\nfetuses\taged\t21+1\tto\t38+2\tgestational\tweeks\t(88\tfemale).\tRepeated\tscans\twere\tacquired\tfrom\tfour\tfetuses.\t\nMRI\tprocessing\t\nWhile\tneonatal\tprotocols\tfor\tautomated\tMRI\ttissue\tsegmentation\texist,87,182\tdue\tto\tthe\tdifferences\tin\tsize,\ttissue\t\ncontrast\tand\tsignal-to-noise\tratio,\tsegmentations\tderived\tfrom\tfetal\tMRI\toften\trequire\textensive\tmanual\tediting\tto\t\nensure\taccuracy.183\t\t\nHere,\twe\tused\tan\toptimised\tneonatal\ttissue\tsegmentation\tpipeline\t(Draw-EM)87\twith\ttissue\tpriors\tadapted\tto\ta\tfetal\t\nMRI\ttemplate\tto\tcreate\ta\t‘first-pass’\ttissue\tsegmentation\tfor\teach\tfetal\tMRI\tvolume.\tTissue\tsegmentations\twere\tthen\t\nvisually\tchecked\tand\textensive\tmanual\tcorrections\tperformed\twhere\tneeded\tto\tcorrect\tgross\tsegmentation\terrors\tand\t\nensure\taccuracy\tof\ttissue\tboundaries\t(CSF/cortex/white\tmatter).\tManually-corrected\ttissue\tsegmentations\twere\tthen\t\nused\tto\tgenerate\tanatomically\tand\ttopologically\tcorrect\tinner\tand\touter\tcortical\tsurfaces\tusing\tDeformable.88\tNote\tthat\t\nall\tintensity-based\tcorrection\tterms\twere\tturned\toff\tduring\tsurface\treconstruction\tand\teach\tsurface\twas\tgenerated\t\nusing\tjust\tthe\tcorrected\ttissue\tsegmentations.\tAt\teach\tstage,\timages\tand\tderived\toutputs\twere\tvisually\tinspected\tfor\t\naccuracy.\t\t\nAlignment\tto\tfetal\ttemplate\t\t\nWe\taligned\tindividual\tcortical\tsurfaces\tto\tthe\tdHCP\tfetal\tatlas,\ta\tspatiotemporal\tsurface\tatlas,\tspanning\t21-36\tweeks\tof\t\ngestation\twith\tweekly\ttimepoints.89,91\tUsing\tMSM\twith\thigher-order\tclique\treduction,\twe\tcalculated\tnon-linear\t\ntransforms\tof\tindividual\tsurfaces\tto\ttheir\tclosest\tfetal\ttimepoint\tbased\ton\tspherical\tregistration\tof\tsulcal\tdepth\t\nfeatures.90,92\tThe\tMSM\ttransform\twas\tused\tto\tresample\tindividual\tsurface\ttopology\t(pial,\tmidthickness,\tand\twhite)\tonto\t\nthe\ttemplate\tsurface\tvertices,\tensuring\tthat\tall\tsurfaces\tacross\tindividuals\thad\tthe\tsame\tvertex\tcorrespondence.\t\nResampled\tsurfaces\twere\tmanually\tchecked\tto\tensure\tthe\tquality\tof\tthe\tregistration.\t\nAlignment\tto\tμBrain\t\nWe\taligned\tthe\tμBrain\tcortical\tsurface\tto\tthe\tearliest\ttimepoint\tof\tthe\tdHCP\tfetal\ttemplate\tsurface\tusing\ta\ttwo-step\t\nnonlinear\tsurface\tregistration\tguided\tby\ta\tset\tof\tanatomical\tpriors\t(Figure\tS17b,c).\tWe\tused\tMSM\tto\tperform\tan\tinitial\t\nnonlinear\tspherical\tregistration\tbetween\tμBrain\tand\tdHCP\tsurfaces\tbased\ton\talignment\tof\tsulcal\tdepth.\tAfter\tthis,\twe\t\ncreated\ta\tset\tof\tcoarse\tcortical\tlabels\ton\tthe\tdHCP\tsurface\tmatched\tto\tcorresponding\tμBrain\tlabels\tby\tcombining\ta)\t\ndHCP\tcortical\tatlas\tlabels,87\tb)\tmanual\tlabels\tguided\tby\tsulcal\tanatomy\ton\tthe\t36\tweek\tfetal\tsurface\tand\tc)\tcombining\t\nμBrain\tlabels\tin\tthe\tsame\tlobes\t(e.g.:\tventrolateral\tfrontal,\tdorsolateral\tfrontal,\torbitofrontal)\twere\tinto\tsingle\t\nanatomical\tlabels.\tThe\tfull\tlist\tof\t11\tmatched\tcortical\tregions\tincluded:\tauditory\tcortex;\tcingulate\tcortex;\tfrontal\tcortex;\t\ninsular\tcortex;\tprimary\tmotor;\tprimary\tsensory;\toccipital\tcortex;\tparahippocampal\tcortex;\tparietal\tcortex;\tsuperior\t\ntemporal\tcortex;\tventrolateral\ttemporal\tcortex.\tA\tsecondary\tmultivariate\tspherical\tregistration\tbetween\tμBrain\tand\t\nfetal\tsurfaces\twas\tinitialised\tusing\tthe\tpreviously\tcalculated\tsulcal\talignment\tand\tdriven\tby\talignment\tof\tcortical\tROIs\t\nacross\tsurfaces.90\tThis\tapproach\tleverages\tanatomical\tlabels\t(defined\tbased\ton\tcytoarchitecture,\tor\tusing\tolder\tfetal\t\nanatomy\tin\tμBrain\tand\tdHCP\tatlases,\trespectively),\tto\tinform\tcortical\talignment\tin\tthe\tabsence\tof\tgeometric\tfeatures.\tA\t\nsimilar\tapproach\thas\tproven\tsuccessful\taccommodating\tlarge\tdeformations\tacross\tprimate\tspecies.184\t\nμBrain\tlabels\twere\tpropagated\tto\teach\ttimepoint\tof\tthe\tdHCP\tfetal\tatlas\t(Figure\t3b)\tand\tonto\tthe\tsurface\ttopology\tof\t\neach\tfetal\tscan.\tCortical\tlabelling\twas\tvisually\tquality\tchecked\tfor\talignment.\t\nStatistical\tanalysis\t\nAllometric\tscaling\tof\tcortical\tsurface\tarea\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n20 \nEach\tsubject’s\touter\tcortical\ttopology\twas\tresampled\tonto\tthe\tdHCP\ttemplate\tsurface\t(32,492\tvertices)\tand\tvertex-wise\t\nestimates\tof\tcortical\tsurface\tarea\twere\tcorrected\tfor\tfolding\tbias\tby\tregressing\tout\tcortical\tcurvature185,186\tand\t\nsmoothed\twith\ta\tGaussian\tkernel\t(FWHM\t=\t10mm).\tTotal\tcortical\tsurface\tarea\twas\tcalculated\tas\tthe\tsum\tof\tall\tvertices\t\nin\tthe\tcortical\tmesh,\texcluding\tthe\tmedial\twall.\tAt\teach\tvertex,\t𝑣,\twe\tmodeled\tscaling\trelationships\twith\tbrain\tsize\tby\t\nestimating\tthe\tlog-log\tregression\tcoefficient\tfor\ttotal\tsurface\tarea\tas\ta\tpredictor\tof\tvertex\tarea,\t𝑎\":42\t\t\n𝑙𝑜𝑔!#(𝑎\") = \t1 + 𝛽𝑙𝑜𝑔!#(8 𝑎\n$\n\"%!\n) \t + 𝜀\t\nSuch\tthat\tthe\tscaling\tcoefficient,\t𝛽,\tcan\tbe\tdirectly\tinterpreted\trelative\tto\t1\t(representing\tlinear\tscaling\tbetween\tvertex\t\narea\tand\ttotal\tarea)\twith\t𝛽 > 1\tand\t𝛽 < 1\trepresenting\thyper-\tand\thypoallometric\tscaling\tof\tvertices\twith\trespect\tto\t\ntotal\tarea,\trespectively.\tModels\twere\tfit\tusing\tOrdinary\tLeast\tSquares\t(OLS)\tregression.\tWe\ttested\talternative\tmodels\t\nincluding\tsex\tand\tage:sex\tinteractions.\t\tAnalyses\twere\trepeated\tafter\tremoving\trepeated\tscans\tto\tsatisfy\ti.i.d.\t\nassumptions\tof\tOLS\tregression\t(n=190;\tFigure\tS18).\t\nPrior\tto\tanalysis,\tvertexwise\toutliers\twere\tidentified\tand\tremoved\t(Figure\tS19).\tTo\taccount\tfor\tage-related\tincreases\tin\t\narea,\toutliers\twere\tidentified\tusing\ta\tsliding\twindow\tover\tage\t(outliers\t>2.5\tS.D.\tfrom\tthe\tmean\twithin\ta\tgiven\twindow,\t\nmaximum\twindow\tsize=25\tscans,\tsorted\tby\tage).\tData\tfrom\tfive\tscans\twere\tremoved\tprior\tto\tanalysis\tdue\tto\tthe\t\npresence\tof\toutliers\tin\tmore\tthan\t5%\tof\tvertices.\t\nVertexwise\tmaps\tof\tareal\tscaling\t(𝛽\tcoefficients)\twere\tparcellated\tusing\tthe\tμBrain\tcortical\tlabels,\tcalculating\taverage\t\nscaling\twithin\teach\tparcel\tfor\tfurther\tanalysis.\t\nModelling\tchanges\tin\tgene\texpression\tover\tzone\t(Z),\tregion\t(R)\tand\ttime\t(T)\t\nFor\teach\tgene\t(n=8771),\twe\tmodelled\tthe\tmain\teffects\tof\tcortical\ttissue\tzone,\tregion\tand\ttimepoint\ton\texpression\tusing\t\na\tgeneral\tlinear\tmodel.\tSignificant\teffects\t(p<0.01)\twere\tidentified\tafter\tFalse\tDiscovery\tRate\tcorrection\tfor\tmultiple\t\ncomparisons\tover\tgenes.\tStatistical\tanalysis\twas\tperformed\tin\tstatsmodels\t(0.13.5)\t\nEnrichment\tanalyses\t\nFor\tall\tenrichment\tanalyses,\twe\tcalculated\tthe\tenrichment\tratio\tas\tthe\tratio\tof\tthe\tproportion\tof\tgenes-of-interest\t\nwithin\teach\tgeneset/marker\tlist\tto\tthe\tproportion\tof\tbackground\tgenes\twithin\teach\tgeneset.\tUnless\totherwise\tstated,\t\nthe\tbackground\tset\twas\tdefined\tas\tthe\tfull\tlist\tof\tgenes\tincluded\tin\tthe\tstudy\t(n=8771).\tSignificance\twas\tdetermined\t\nusing\tthe\thypergeometric\tstatistic:\t\n𝑝 = 1 − 8\n>𝐾\n𝑖 A >𝑀 − 𝐾\n𝑁 − 𝑖 A\n>𝑀\n𝑁A\n&\n'%#\n\t\nWhere\tp\tis\tthe\tprobability\tof\tfinding\tx\tor\tmore\tgenes\tfrom\ta\tspecific\tgeneset\tK\tin\ta\tset\tof\trandomly\tselected\tgenes,\tN\t\ndrawn\tfrom\ta\tbackground\tset,\tM.\tWhere\tstated,\tFalse\tDiscovery\tRate\t(FDR)\tcorrection\twas\tapplied\tto\tmultiple\t\ncomparisons.\t\nCode\tand\tdata\tavailability\t\nThe\tμBrain\tdigital\ttemplate\twith\tcorresponding\tcortical\tsurfaces\tand\tatlas\tlabels\tis\tavailable\tfrom\t\nhttps://garedaba.github.io/micro-brain\talongside\tcode\tsupporting\tdata\tprocessing\tand\tanalysis\tfor\tthis\tmanuscript.\t\nAll\tdHCP\tdata,\tfetal\tbrain\treconstructions,\tbrain\tregion\tsegmentations\tand\tcortical\tsurfaces\tare\tavailable\tfor\tdownload\t\nfrom\tthe\tNDA\thttps://nda.nih.gov/edit_collection.html?id=3955\t\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint \n\n21 \nSource\thistological\tand\tmicroarray\tdata\tare\tavailable\tfrom\tthe\tAllen\tBrain\tInstitute\thttps://www.brainspan.org/\t\n\t\nReferences\t\n\t\n1.\t Polioudakis,\tD.\tet\tal.\tA\tSingle-Cell\tTranscriptomic\tAtlas\tof\tHuman\tNeocortical\tDevelopment\tduring\tMid-gestation.\t\nNeuron\t103,\t785-801.e8\t(2019).\t\n2.\t Bhaduri,\tA.\tet\tal.\tAn\tatlas\tof\tcortical\tarealization\tidentifies\tdynamic\tmolecular\tsignatures.\tNature\t598,\t200–204\t\n(2021).\t\n3.\t Ding,\tS.-L.\tet\tal.\tCellular\tresolution\tanatomical\tand\tmolecular\tatlases\tfor\tprenatal\thuman\tbrains.\tJ.\tComp.\tNeurol.\t\n530,\t6–503\t(2022).\t\n4.\t Miller,\tJ.\tA.\tet\tal.\tTranscriptional\tlandscape\tof\tthe\tprenatal\thuman\tbrain.\tNature\t508,\t199–206\t(2014).\t\n5.\t García-Cabezas,\tM.\tÁ.,\tZikopoulos,\tB.\t&\tBarbas,\tH.\tThe\tStructural\tModel:\ta\ttheory\tlinking\tconnections,\tplasticity,\t\npathology,\tdevelopment\tand\tevolution\tof\tthe\tcerebral\tcortex.\tBrain\tStruct.\tFunct.\t224,\t985–1008\t(2019).\t\n6.\t Brodmann,\tK.\tVergleichende\tLokalisationslehre\tder\tGrosshirnrinde.\t(Barth,\t1909).\t\n7.\t Sanides,\tF.\tThe\tcyto-myeloarchitecture\tof\tthe\thuman\tfrontal\tlobe\tand\tits\trelation\tto\tthe\tphylogenetic\t\ndifferentiation\tof\tthe\tcerebral\tcortex.\tJ.\tHirnforsch.\t7,\t269–282\t(1964).\t\n8.\t Petrides,\tM.\t&\tPandya,\tD.\tN.\tDorsolateral\tprefrontal\tcortex:\tcomparative\tcytoarchitectonic\tanalysis\tin\tthe\thuman\t\nand\tthe\tmacaque\tbrain\tand\tcorticocortical\tconnection\tpatterns.\tEur.\tJ.\tNeurosci.\t11,\t1011–1036\t(1999).\t\n9.\t Barbas,\tH.\t&\tRempel-Clower,\tN.\tCortical\tstructure\tpredicts\tthe\tpattern\tof\tcorticocortical\tconnections.\tCereb.\t\nCortex\t7,\t635–646\t(1997).\t\n10.\t Economo,\tC.\tvon\t&\tKoskinas,\tG.\tN.\tDie\tCytoarchitektonik\tder\tHirnrinde\tdes\terwachsenen\tMenschen.\t(J.\tSpringer,\t\n1925).\t\n11.\t Rakic,\tP.\tSpecification\tof\tcerebral\tcortical\tareas.\tScience\t241,\t170–176\t(1988).\t\n12.\t Letinic,\tK.,\tZoncu,\tR.\t&\tRakic,\tP.\tOrigin\tof\tGABAergic\tneurons\tin\tthe\thuman\tneocortex.\tNature\t417,\t645–649\t\n(2002).\t\n13.\t Kriegstein,\tA.\tR.\t&\tNoctor,\tS.\tC.\tPatterns\tof\tneuronal\tmigration\tin\tthe\tembryonic\tcortex.\tTrends\tNeurosci.\t27,\t392–\n399\t(2004).\t\n14.\t O’Leary,\tD.\tD.,\tSchlaggar,\tB.\tL.\t&\tTuttle,\tR.\tSpecification\tof\tneocortical\tareas\tand\tthalamocortical\tconnections.\t\nAnnu.\tRev.\tNeurosci.\t17,\t419–439\t(1994).\t\n15.\t Elsen,\tG.\tE.\tet\tal.\tThe\tprotomap\tis\tpropagated\tto\tcortical\tplate\tneurons\tthrough\tan\tEomes-dependent\tintermediate\t\nmap.\tProc.\tNatl.\tAcad.\tSci.\t110,\t4081–4086\t(2013).\t\n16.\t Ypsilanti,\tA.\tR.\tet\tal.\tTranscriptional\tnetwork\torchestrating\tregional\tpatterning\tof\tcortical\tprogenitors.\tProc.\tNatl.\t\n.CC-BY 4.0 International licenseperpetuity. 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It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 13, 2024. ; https://doi.org/10.1101/2024.02.13.580198doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}