22q11 deletion selectively alters progenitor states and projection neuron identities in the developing cerebral cortex

22q11 deletion selectively alters progenitor states and projection neuron identities in the developing cerebral cortex | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results 22q11 deletion selectively alters progenitor states and projection neuron identities in the developing cerebral cortex View ORCID Profile Shah Rukh , Daniel W. Meechan , Connor Siggins , Zachary D. Erwin , Gia Baldo , Ashley Peck , View ORCID Profile Thomas M. Maynard , View ORCID Profile Anthony-Samuel LaMantia doi: https://doi.org/10.1101/2025.05.12.653556 Shah Rukh 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; 2 Translational Biology, Medicine and Health Program , Virginia Tech, Blacksburg, VA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shah Rukh Daniel W. Meechan 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Connor Siggins 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zachary D. Erwin 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gia Baldo 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashley Peck 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas M. Maynard 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas M. Maynard Anthony-Samuel LaMantia 1 Center for Neurobiology Research, The Fralin Biomedical Research Institute at Virginia Tech-Carilion School of Medicine , Roanoke, VA; 3 Department of Biological Sciences , Virginia Tech, Blacksburg, VA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anthony-Samuel LaMantia For correspondence: anthonysl{at}vtc.vt.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Heterozygous deletion of multiple contiguous genes associated with 22q11.2 Deletion Syndrome (22q11DS), a developmental disorder with significant risk for autistic spectrum disorder (ASD) and schizophrenia (Scz), selectively compromises neurogenic capacities of a temporally distinct cohort of cerebral cortical basal progenitors (bPs), prefiguring diminished frequency, divergent times of origin, positions, and identities of a subset of Layer 2/3 projection neuron (PN) progeny in the LgDel 22q11DS mouse model. LgDel bPs express 24/28 contiguous murine 22q11 gene orthologues at diminished levels; in parallel, cell cycle kinetics, modes of division, gene expression levels, and DNA methylation states are aberrant in LgDel bPs but not their apical progenitor precursors. Accordingly, targeted disruption of bP proliferative and transcriptional states selectively alters Layer 2/3 PN identities and frequencies, prefiguring atypical association cortico-cortical connections and behavioral deficits associated with ASD and Scz pathology in a mouse model of 22q11DS. INTRODUCTION Disrupted projection neuron (PN) genesis in the cerebral cortex is thought to underlie pathology in a broad range of neurodevelopmental disorders (NDDs), including Autistic Spectrum Disorder (ASD) and Schizophrenia (Scz) 1 – 3 . Nevertheless, it is unclear whether or how PN neurogenic mechanisms that end long before the onset of NDD behavioral pathology can be compromised by polygenic changes thought to lead to disrupted cortical circuit function in most NDDs 4 , 5 . NDD pathogenesis may target specific cortical progenitor types, especially those that generate upper layer (Layer 2/3) PNs essential for association cortico-cortical connections thought to be compromised in multiple NDDs. Thus, we asked whether polygenic changes underlying 22q11.2 Deletion Syndrome, a copy number variant (CNV) disorder with one of the highest known genetic associations with Scz and ASD risk, broadly or selectively compromise cortical precursors that generate the majority of Layer 2/3 PNs. Over the past 2 decades, analyses of PN neurogenesis have focused on two lineally related precursor types: apical progenitors (aPs): slowly dividing stem cells at or near the ventricular zone (VZ), and basal progenitors (bPs): rapidly dividing aP-derived intermediate precursors in the adjacent subventricular zone (SVZ), that generate primarily Layer 2/3 PNs 6 – 8 . Our previous work in LgDel 22q11DS model mice 9 identified reduced bP proliferation as an antecedent of diminished postnatal Layer 2/3 PN frequency due to 22q11 gene deletion 10 . Nevertheless, the relationship of this change to altered molecular identities, lineage progression, or cellular states of bPs, their aP precursors, and neuroblast (NB) progeny due to diminished expression of multiple contiguous 22q11-deleted genes remains unknown. Diminished dosage of one, several, or all 22q11 genes may broadly or selectively influence aP or bP neurogenic capacity, NB differentiation and subsequent Layer 2/3 PN maturation 11 , 12 . Such changes may compromise Layer 2/3 PNs proportionately by uniformly slowing aP or bP proliferation, yielding fewer progeny whose birthdates and identities nevertheless parallel wild type (WT). Alternately, 22q11 deletion may result in temporal- or cell type-selective changes that disproportionately alter neurogenic capacities of distinct aP or bP cohorts and differentiation trajectories for subsets of Layer 2/3 PNs. Based upon these unanswered questions, including previous observations of altered PN differentiation and function in LgDel mice 10 , 13 , 14 , we asked whether 22q11 deletion disrupts cortical development by selectively modifying proliferative kinetics, identities, or transcriptional and cell biological states of aPs, bPs or their Layer 2/3 PN progeny. We used quantitative cellular assays, transcriptional profiling, neuronal birth-dating and canonical molecular markers of progenitor or PN identity in LgDel versus WT fetal or early postnatal frontal association cortex to assess selectivity and timing of 22q11 deletion’s consequences on Layer 2/3 PN genesis. 22q11 deletion selectively disrupts bP, but not aP, neurogenic capacity and transcriptional states during peak Layer 2/3 PN genesis without loss or gain of bP or NB subtypes. Instead, 22q11 gene dosage alters dynamic bP/NB states. Evidence of diverse states includes: SVZ cells that express multiple bP, NB and Layer 2/3 PN markers while retaining proliferative capacity; variable per cell expression of 22q11 genes as well as others that modulate neurogenesis, and bP-selective DNA methylation changes in a multigene locus associated with neurogenesis. These bP-targeted changes prefigure generation of a diminished Layer 2/3 PN cohort with divergent times of genesis, molecular identities, and positions—long before emergence of dysfunctional cortical circuits thought to underlie NDD pathology. RESULTS 22q11 deletion selectively compromises bP proliferative capacity We first asked whether 22q11 deletion uniformly or selectively alters aP to bP to NB lineage progression as Layer 2/3 PN genesis accelerates between embryonic day (E) 13.5 and 15.5 10 , 15 , prefiguring diminished postnatal LgDel association cortical Layer 2/3 PN frequency 10 , 13 . We focused on a standard cortical location, slightly lateral in the frontal pole, anterior to the ganglionic eminences. At E13.5, when aPs accelerate bP generation, distribution of Pax6 + aPs, Pax6 + /Tbr2 + presumed newly generated bPs among the Tbr2 + bP population does not differ noticeably in LgDel vs. WT VZ, SVZ or intermediate zone (IZ; Figure 1a, b ), nor does their frequency ( Figure 1c ; see also Figure 2 ). Apparently, based upon analysis of cells expressing canonical markers, 22q11-deleted aPs generate bPs at similar frequencies to WT as the first bP cohort emerges by E13.5, immediately prior to peak Layer 2/3 PN genesis. Download figure Open in new tab Figure 1: 22q11 deletion disrupts bP, but not aP, proliferation during Layer 2/3 PN genesis. a, b ). E13.5 WT ( a ) and LgDel ( b ) cortical apical and basal progenitors (aPs: Pax6 + ; bPs: Tbr2 + ). aPs predominate in the ventricular zone (VZ), and bPs in the subventricular zone (SVZ). Pax6 + /Tbr2 + presumed newly generated bPs (arrows, hi-mag. ) are seen primarily at the VZ/SVZ border. c ). Equivalent frequencies of Pax6 + aPs and Pax6 + /Tbr2 + bPs in the 2 genotypes; numbers (n) of fetuses analyzed, and p-values (unpaired t-tests) shown in each histogram. d, e ). E14.5 WT ( d ) and LgDel ( e ) Tbr2 + bPs, Tbr2 + /Neurod1 + presumed newly generated Layer 2/3 neuroblasts (NBs), and Neurod1 + presumed maturing/migrating cortical NBs; bPs mostly in the SVZ; new NBs at the SVZ/intermediate zone (IZ) interface (arrows, hi-mag. panels ), and maturing NBs in the IZ and cortical plate (CP). f ). Tbr2 + cell frequency is indistinguishable in WT and LgDel ( top ); however, Tbr2 + /Neurod1 + NB frequency increases significantly ( bottom ). g, h ). E15.5 presumed WT ( g ) and LgDel ( h ) Cux1-2 + Layer 2/3 projection neurons (PNs). Cux1-2 + cell frequency appears diminished in LgDel CP ( top , high mag. panels ). Presumed migrating Cux1-2 + Layer 2/3 PNs and Tbr2 + / Cux1-2 + cells are apparently similarly frequent in the IZ ( middle , high mag .). Tbr2 + cells predominate in the SVZ, accompanied by occasional Cux1-2 + cells in the VZ and SVZ ( bottom , high mag .). i ). Cux1-2 + presumed Layer 2/3 PNs decrease significantly in E15.5 LgDel cortex ( top ), with no change in Tbr2 + /Cux1-2 + cell frequency ( middle ). E13.5 and 14.5 Cux1-2 + cell frequency is low, and statistically indistinguishable in WT and LgDel , but declines significantly by E15.5 (2-way ANOVA with Šídák’s multiple comparisons test, bottom ). Download figure Open in new tab Figure 2: 22q11 deletion alters bP cell cycle re-entry and neurogenic division frequency a ). Distributions of E13.5 EdU + (S-phase, E12.5 maternal EdU injection, blue ) WT ( left ) and LgDel ( middle ) Pax6 + ( green )/ Ki67 + (proliferative marker, red ) aPs. inset : equivalent WT vs. LgDel Pax6 + cell frequency ( middle , unpaired t-test). WT (arrows, right , top ) and LgDel (arrows, right , bottom ) EdU + /Pax6 + / Ki67 + cells represent aPs that retain capacity to re-enter the cell cycle. b ). E13.5 WT and LgDel Pax6 + /EdU + /Ki67 + aP frequencies are equivalent (unpaired t-test). c ). E15.5 WT ( left ) and LgDel ( right ) EdU + (E14.5 maternal EdU injection, blue )/ Tbr2 + ( green )/ Ki67 + ( red ). WT and LgDel bPs that presumably re-enter the cell cycle (arrow, right, top & bottom ). inset : Tbr2 + cells decrease significantly in E15.5 LgDel ( middle , unpaired t-test). d ). E15.5 Tbr2 + /EdU + /Ki67 + bP frequency declines significantly in LgDel (unpaired t-test). e ). Tbr2 + bP frequency does not decline in LgDel cortex until E15.5 (independent E13.5, 14.5 & 15.5 samples; n’s in histogram bars; 2-way ANOVA with Šídák’s multiple comparisons test). f ). Isolated pairs (DAPI + , blue ) of in vitro presumed bPs (Tbr2 + , green ) undergoing symmetric (2 Tbr2 + ) or asymmetric (1 Tbr2 + , 1 Neurod1 + , red ) self-renewing vs. terminal neurogenic division (2 Neurod1 + ). g ). Frequency of symmetric (dark green), asymmetric (light green) and terminal neurogenic divisions (red) among WT and LgDel presumed in vitro bPs at E12.5, 14.5 and 16.5. At each age, self-renewing (symmetric/asymmetric) divisions decline and terminal neurogenic divisions increase significantly among LgDel bPs (Chi-square; E12.5: 6 WT, 6 LgDel fetuses/2 litters; E14.5: 14 WT, 14 LgDel fetuses/5 litters; E16.5: 11 WT, 10 LgDel fetuses/4 litters). To assess whether 22q11 deletion alters E14.5 bP neurogenic capacity, we compared the distribution and frequency of LgDel vs. WT Tbr2 + bP and Tbr2 + /Neurod1 + presumed newly generated NBs. E14.5 WT and LgDel Tbr2 + bPs are seen across the upper VZ, SVZ and lower IZ, and their WT vs. LgDel frequencies are indistinguishable ( Figure 1d-f ; see also Figure 2e ). In contrast, LgDel Tbr2 + /Neurod1 + NB frequency increases significantly in the upper SVZ/IZ without concomitant decrease of Tbr2 + cells ( Figure 1e, f ) or increase of Neurod1 + cells ( not shown ). Thus, as Layer 2/3 PN genesis accelerates at E14.5, 22q11-deleted bPs produce more NBs— identified by canonical markers—without a change in total bP frequency. If the increase in newly generated NBs at E14.5 reflects premature neurogenesis that exhausts an early bP cohort to diminish Layer 2/3 PN genesis thereafter, E15.5 Cux1-2 + presumed early differentiating LgDel Layer 2/3 PN frequency should increase, reflecting the surfeit of E14.5-generated NBs. The distribution of Cux1-2 + presumed Layer 2/3 PNs as well as Tbr2 + bPs is not noticeably changed in LgDel E15.5 fetuses ( Figure 1g, h ); however, LgDel Cux1-2 + cell frequency declines significantly in the IZ and CP ( Figure 1h, i ; top ). Thus, based upon canonical markers, 22q11-deleted E14.5 Tbr2 + bPs generate an ambiguous subset of SVZ cells with that do not contribute immediately to the E15.5 LgDel Cux1-2 + Layer 2/3 PN population. Divergent cell cycle kinetics and modes of division in 22q11-deleted bPs Increased E14.5 LgDel NBs without subsequently increased Layer 2/3 PN frequency suggests that 22q11 deletion may alter progenitor cell cycle dynamics and modes of division. To assess proliferative kinetics in 22q11-deleted aPs and bPs as Layer 2/3 PNs are generated, we analyzed cell cycle exit vs. re-entry ( Figure 2a-d ) using combined S-phase/Ki67 labeling 16 . Distributions and frequencies of E13.5 Pax6 + /EdU + /Ki67 + aPs—which presumably can re-enter the cell cycle— are similar in LgDel vs. WT ( Figure 2 a,b ), as are Pax6 + cell frequencies ( Figure 2a , inset ). In contrast, frequency of E14.5 LgDel bPs that retain the capacity to re-enter the cell cycle by E15.5 declines ( Figure 2c, d ). In parallel, there are fewer E15.5 Tbr2 + cells in LgDel ( Figure 2c , inset ). To resolve stability vs. change in LgDel vs. WT Tbr2 + bP cohorts, we analyzed E13.5, E14.5 and E15.5 Tbr2 + cell frequency in a separate sample of non-EdU injected fetuses. There are significantly fewer LgDel vs. WT E15.5—but not E13.5 or 14.5—Tbr2 + bPs ( Figure 2e ). Thus, within the E14.5 22q11-deleted bP cohort, cell cycle kinetics change, favoring apparent NB identity; however, stability of Tbr2 + cell frequency and parallel decrease of E15.5 Layer 2/3 PNs (see Figure 1g-i ) and suggests that some 22q11-deleted NBs remain in a liminal state. Divergent cell cycle kinetics may bias 22q11-deleted bP divisions toward terminal neurogenesis. We used an in vitro pair cell assay to assess the capacity of isolated LgDel vs. WT bPs to undergo symmetric or asymmetric self-renewing vs. symmetric neurogenic divisions at E12.5 when bPs are few, E14.5 when a robust bP cohort emerges, and E16.5 as the bP population declines. Most WT and LgDel Tbr2 + bPs in vitro yield two Neurod1 + NBs ( Figure 2f ); however, LgDel symmetric or asymmetric self-renewing bP divisions decline significantly and terminal neurogenic divisions increase ( Figure 2g ). The shift from self-renewal to terminal neurogenesis is consistent at all ages with some variation of symmetric vs. asymmetric self-renewing divisions ( Figure 2g ). Apparently, 22q11 deletion selectively disrupts bP cell cycle dynamics and self-renewal during peak Layer 2/3 PN genesis to accelerate yield of apparent NBs; however, the fates of these surplus 22q11-deleted NBs are not clear. 22q11 deletion alters transcriptional states—but not identities—of bPs and NBs Our cell biological analyses suggests that 22q11 deletion alters bP or NB characteristics not captured by canonical bP, NB, PN or proliferative markers. To determine whether there are divergent LgDel vs. WT bP or NB subtypes that cannot be identified by combinations of cardinal markers, we performed single cell RNA sequencing (scRNAseq) in E14.5 LgDel and WT cortical cells ( Figure 3a ). We compared 4772 WT and 3272 LgDel cells (50,000 reads/cell): ∼75% from 5 WT and 3 LgDel E14.5 cortices; ∼25% from 5 WT and 7 LgDel Tbr2 eGFP+ cell-sorted samples of presumed bPs and their immediate progeny 17 . An unbiased UMAP analysis of all cells (unsorted and Tbr2 eGFP+ ) identifies small, discrete interneuron, endothelial, glia/tanycytes, and undifferentiated neuroepithelial cell clusters ( Figure 3a , top ). The largest clusters comprise two Pax6/Nes + / Fabp7 + / Slc1a3 + aP subsets within a single domain and a continuous aggregate of three bP subsets that express higher levels of Tbr2 + , Tis21 + and Neurog2 + adjacent to six Neurod1 + /Dcx + / Tubb3 + NB subsets ( Figure 3a , middle ; Figure 3b ). No aP, bP, or NB subsets were lost or added in LgDel ( Figure 3a , bottom ), and equivalent expression of aP, bP or NB associated genes across subsets ( Figure 3b ), reinforces their congruity in E14.5 WT and LgDel cortices. Download figure Open in new tab Figure 3: Similar transcriptional identities for WT and LgDel progenitor and NB subtypes a ). UMAP analysis of single cell RNA sequencing (scRNAseq) of 8044 WT and LgDel E14.5 cortical cells ( top ). Endothelial cells (Endo), tanycytes (Tany), neuroepithelial precursors (nEP) and GABAergic interneurons (IN) cluster discretely. The two largest clusters are an aP aggregate with two subsets (yellow/yellow-green; 1,2 numbered boxes), and a continuous accumulation of bPs and NBs comprising 9 overlapping subsets: 3 bPs (green/numbered boxes 1 - 3) and 6 NBs (blue/numbered boxes 1-6). Pax6 + , Tbr2 + , and Neurod1 + cells ( middle, left to right ) map to regions corresponding to inferred aP, bP or NB subsets. Comparison of WT (non-sorted: 3402 cells) and LgDel (non-sorted: 1993 cells) aP and bP/NB aggregates ( bottom ) shows that all aP, bP and NB subsets are shared by the two genotypes. b ). Confirmation of identity and quantitative stability of additional aP, bP and NB markers across shared WT and LgDel aP, bP and NB subsets. (lighter half-circles: WT; darker half-circles: LgDel ; frequency scale, bottom ). c ). Expression of 15 22q11 genes (threshold: mean 0.1 transcripts/cell) across all aP, bP and NB subsets. Smaller dark half-circle sizes indicate diminished mean LgDel expression levels. Transcriptionally equivalent WT vs. LgDel aP, bP and NB subsets may reflect limited expression or dosage compensation of 22q11 genes. This does not seem to be the case: 15/28 murine 22q11 orthologues are expressed robustly across all aP, bP, and NB subsets in both genotypes ( Figure 3c ). Ranbp1 , Dgcr8 and Cdc45 (progenitor-enhanced 11 , 18 , 19 ) vs. Rtn4r , Tango2 , and Sept5 (neuron-enhanced 19 – 21 ) vary in register with aP, bP and NB identity. Expression of these six, plus nine additional 22q11 genes—including Gnb1L , Dgcr2 , and Comt , independently associated with ASD or Scz risk 22 , 23 —declines by approximately 50% in all LgDel subsets. In parallel, all subsets in both genotypes express microcephaly genes 24 and ASD risk genes 23 at consistent levels; however, ASD risk genes are more highly expressed. In contrast, many high confidence Scz candidates 22 are minimally expressed or absent in aP, bP, and NB subsets ( Supplemental Figure 1 ). Despite this variation across NDD risk gene categories: ASD > microcephaly > Scz; WT and LgDel aP, bP and NB expression levels are indistinguishable. Thus, scRNAseq analysis resolves aP, bP, and NB subsets; however, overlapping distribution and lack of singular markers, including 22q11 or NDD candidate genes, suggest that subset designations reflect quantitative transcriptional or cellular states 25 – 27 rather than distinct cell types. 22q11 deletion influences bP and NB states Diminished expression of multiple 22q11 genes may influence cellular states in otherwise transcriptionally similar WT vs. LgDel aP, bP or NB subsets, disproportionately altering frequency, lineage or cell biological mechanistic features of one or more subsets. Proportions of each scRNAseq defined subset in WT vs. LgDel cortex (corrected for reduced size of E14.5 LgDel vs WT cortex; Figure 4a ) vary. WT and LgDel aP1 and 2 as well as bP1 subsets are indistinguishable. Proportions of bP 2 and 3 as well as four of six NB subsets diverge in LgDel vs. WT, and as a group, all 6 LgDel NB subsets differ ( Figure 4a , bottom right ). To determine if LgDel bP or NB subsets have equivalent proportions of Tbr2 + precursor-derived cells, we mapped the distribution of Tbr2 eGFP+ cells, which include Tbr2 + bPs and their immediate progeny, within aP/bP/NB subsets ( Figure 4b ). Tbr2 expression aligns with eGFP across all subsets except NB6, which may reflect the limited number of cells in this subset in WT and LgDel ( Figure 4b , middle , left ). Tbr2 eGFP+ cells are distributed across WT and LgDel bP and NB subsets uniformly ( Figure 4b , top right ) with approximately equivalent frequencies ( Figure 4b , bottom ), suggesting that no Tbr2 + -expressing or -derived subset is disproportionately altered by 22q11 deletion. Download figure Open in new tab Figure 4: Quantitative divergence of LgDel bP and NB transcriptional and cell states a ). E14.5 WT ( top , lighter) and LgDel ( bottom , darker) aP, bP, and NB subsets as a proportion of all cells (gray: cells outside aP/ bP/NB aggregates); LgDel normalized for in vivo cortical cell frequency (standard counting frame, arrow-apical to basal; bottom left ). Proportions of WT and LgDel cells in aP1 and 2 as well as bP1 are indistinguishable (per animal, Mann-Whitney; n = 5 WT, 3 LgDel ). LgDel bP2 and 3 proportions decline, and as a group, LgDel NB subsets diverge significantly (Mann-Whitney; bottom , far right ). b ). Distribution of Tbr2 eGFP+ progenitors and NB progeny ( top left ) across aP, bP and NB subsets. Tbr2 and eGFP transcripts detected at approximately equivalent frequencies ( middle left ). Tbr2 eGFP+ cells are rare and distributed similarly to Tbr2 + cells in aP subsets; however, they are uniformly distributed across the bP/NB aggregate at higher frequency than that of Tbr2 + cells ( top right ). WT vs. LgDel Tbr2 and eGFP transcript frequencies are indistinguishable: enriched in bPs, diminished in NB subsets ( middle left ; light vs. dark half circles). c ). Proportions of cells in G2/M (green), S (orange), or G1 (gray), estimated based upon expression levels of canonical cell cycle genes ( top ), in WT and LgDel aP, bP and NB subsets. G2/M and S cells are detected in all aP, bP or NB subsets. They decline significantly in LgDel bP1 and 3 as well as NB 1 and 2, and increase significantly in LgDel NB5 and 6 (per animal, Mann-Whitney; n=5 WT, 3 LgDel ). PCA clusters ( bottom ) of G1 (gray dots), G2/M (green dots) and S (orange dots) cells in WT vs. LgDel aP and NB subsets with no differences (aP1, NB3) or significant differences (bP1, NB6). d ). Canonical upper and lower layer PN markers expressed at similar levels in WT and LgDel aP, bP and NB subsets. The aP, bP and NB subsets were identified after excluding 22q11 genes and cell cycle genes to avoid artifact due to diminished 22q11 transcript frequency or shared proliferative state. Thus, we examined distribution of cells in G1, S, and G2/M of the cell cycle, inferred based upon expression levels of key genes associated with each phase 28 , to determine whether proliferative states diverge in LgDel aP, bP or NB subsets ( Figure 4c ). All LgDel and WT aP, bP and NB subsets have proliferative cells defined by these transcriptional criteria. Substantial proportions of G1, S or G2/M cells are seen in both aP subsets, and decline monotonically in bP1 through 3 ( Figure 4c , left ). Despite presumed neural identity, G2/M and S cells are detected in all NB subsets—at greater frequency in NB2 and 6 than in two of three bP subsets ( Figure 4c , right ). LgDel G2/M and S cell proportions differ from WT; lower in bP1, 3 and NB1, 3; higher in NB5, 6. This suggests that increased newly generated E14.5 NB frequency (see Figure 1 ) reflects diverse proliferative states in cells with NB transcriptional signatures. Robust expression of canonical cortical PN markers across all aP, bP and NB subsets reinforces this ambiguity ( Figure 4d ). There are modest biases in upper vs. lower layer marker expression; however, none are excluded from any subset, nor do any differ in WT vs. LgDel . Apparently, loss or gain of progenitors or NB “types” defined solely by gene expression does not prefigure 22q11 deletion-dependent decline in Layer 2/3 PN genesis. Instead, 22q11 deletion alters cell states including those of “occult” populations of apparent NBs that retain proliferative capacities. Transcriptional divergence in 22q11-deleted Tbr2+ cells scRNAseq comparisons of LgDel vs. WT indicate that 22q11 deletion primarily targets Tbr2 + precursors and their progeny. To better resolve differences between 22q11-deleted vs. WT bPs or NBs, we sorted Tbr2 eGFP+ cells from LgDel and WT E14.5 cortices (see Figure 4b ) for bulk RNA sequencing. In 5 pooled samples/genotype ( Figure 5a , left ) 19645 genes are significantly expressed (≥ 15 read counts) in WT, and 18057 in LgDel ( Figure 5b ). 79 functionally annotated genes were differentially expressed (DE) in LgDel Tbr2 eGFP+ E14.5 cells (FDR < 0.1). A majority— 50—were upregulated, while 29 were downregulated ( Figure 5a, b ). Thirteen upregulated genes, including Col5a2 , Bcan , Dlk1 , Gadd45g and Slit3 , have been associated with cortical neurogenesis ( Figure 5b , inset ). Quantitative PCR from parallel Tbr2 eGFP+ samples confirms upregulation of a subset of these genes ( Supplemental Figure 2 ). Twenty-four 22q11-deleted genes are expressed in WT and LgDel Tbr2 eGFP+ cells (FDR < 0.1), and their LgDel expression declines by ∼50% ( Figure 5b , bottom ). Thus, significant expression of nine 22q11 genes not identified by scRNAseq can be detected in Tbr2 + cells and their immediate progeny. Similarly, 39 of 50 upregulated DE genes were detected across all scRNAseq progenitor and NB subsets; however, bulk RNAseq expression differences were not consistently apparent ( Supplemental Figure 3 ). In parallel, DE and 22q11 genes, assessed using the GenePaint database 28 , can be detected in the VZ, SVZ, IZ, CP or MZ, and several are enhanced in the SVZ ( Supplemental Figure 4 ). Thus, transcriptional identities of 22q11-deleted Tbr2 + cells and their immediate progeny are further resolved by bulk RNAseq analysis of multiple pooled biological replicate samples. Download figure Open in new tab Figure 5: Differentially expressed genes in bPs and their progeny a ). Heatmaps of differentially expressed (DE) genes (FDR < 0.1) in WT and LgDel Tbr2 eGFP+ -sorted cells across 5 pooled biological replicates ( left ). Normalized enrichment scale (WT vs. LgDel ): between 1-fold (yellow) and zero-fold (blue). 22q11 genes are highlighted (orange; left margin). Summary heatmap ( right ) of average TPMs for DE genes: fold differences ≥ +1.5 (yellow) through −1.5 (blue). b ) Venn diagram of DE genes as a subset of all transcripts (FDR < 0.1) in WT vs LgDel Tbr2 eGFP+ cells ( top left ). A volcano plot of DE genes (p < 0.01 dotted line; top right ) shows the fairly consistent 50% decrease (−1 log fold) for 22q11 genes (orange, left ), and a range of increased expression for additional DE genes ( right ) including a subset of 13 associated with cortical neurogenesis ( inset ). Expression of 24 murine 22q11.2 orthologues (FDR < 0.1) in LgDel Tbr2 eGFP+ cells is reduced to approximately 50% of WT levels (dotted line; bottom ). c ). Gene set enrichment analysis (GSEA) of hallmark pathways in LgDel (p.adj < 0.05): positively enriched, upper right; negatively enriched, lower left. Pink (high)-to-blue (low) scale indicates statistical significance (p-adjust). Circle sizes indicate gene numbers (Count) in each hallmark gene set. We next asked if in LgDel DE genes are associated with broader signaling or cell state pathways in 22q11-deleted Tbr2 + bPs and their immediate progeny. Across 50 hallmark gene sets 29 ( Supplemental Figure 5 ), there is significant positive enrichment (p<0.05) of eight sets, including proliferation, cell cycle, and cytoskeleton pathways ( Figure 5c , top ). Enriched G2M checkpoint and mitotic spindle hallmarks suggest divergent proliferative activity, consistent with aberrant LgDel bP and NB proliferative characteristics; epithelial-mesenchymal transition pathways may reflect retention or delayed migration of LgDel NBs in ambiguous states; KRAS pathways, up or down, may indicate 22q11-deletion dependent signal transduction changes that modify bP or NB capacities. Four negatively-enriched hallmarks: oxidative phosphorylation, Myc Targets, DNA repair and MTORC1 ( Figure 5c , bottom ) suggest bioenergetic dysfunction in 22q11-deleted bPs or NBs, potentially due to inefficient mitochondrial ROS clearance 14 or an anomalous metabolic shift toward glycolysis 30 , 31 . These statistical indications of altered transcription associated with key cell biological mechanisms in LgDel Tbr2 eGFP+ cells are consistent with divergent cell states across subsets of 22q11-deleted bPs and their NB progeny that disrupt neurogenic capacity to diminish Layer 2/3 PN frequency. Heterogeneous transcriptional states of 22q11-deleted bPs Neither scRNAseq nor bulk RNAseq resolves whether 22q11 or DE gene expression changes uniformly or selectively in individual cortical SVZ/IZ progenitors or neighboring cells. Thus, we analyzed quantitative transcription of select 22q11 and DE genes in individual E14.5 LgDel or WT Tbr2 + or Tbr2 - /DAPI + SVZ/IZ cells ( Figure 6 ). Cdc45 and Dgcr8 transcripts per cell, based upon RNAscope labeling ( Figure 6a, b ), in Tbr2 + and Tbr2 - /DAPI + SVZ/IZ cells are not normally distributed ( Figure 6c , Supplemental Figure 6 ). Instead, Poisson distributions for each reach a WT maximum at 1 Cdc45 punctum and 5 Dgcr8 puncta/cell vs. 1 Cdc45 punctum and 2 Dgcr8 puncta/cell for LgDel . LgDel Tbr2 + and Tbr2 - cells with no Cdc45 or Dgcr8 puncta increase, while those with higher puncta frequencies decline ( Figure 6c , Supplemental Figure 6 ). Mean Cdc45 and Dgcr8 puncta frequency in Tbr2 + and Tbr2 - cells diminishes by approximately 50% ( Figure 6c , insets ; Supplemental Figure 6 ); however, levels are not substantially correlated in individual Tbr2 + or Tbr2 - cells ( Figure 6c , bottom ; Supplemental Figure 6 ), and are independent of cell size ( Supplemental Figure 6 ). Variability and autonomy of 22q11 gene expression levels in individual Tbr2 + and Tbr2 - cells is consistent with a spectrum of transcriptional states that may differentially influence 22q11-deleted vs. WT bP or NB proliferative and neurogenic capacities. Download figure Open in new tab Figure 6: 22q11 deletion disrupts transcriptional variation in individual cortical progenitors a ). Broad range (1-10) of Cdc45 RNAscope puncta (violet) in WT or LgDel Tbr2 + VZ/SVZ cells. b ). Dgcr8 RNAscope puncta/Tbr2 + VZ/SVZ cell (red) do not appear highly correlated with Cdc45 (violet) in WT or LgDel . c ). Cdc45 puncta frequency reaches a maximum at 1 punctum/ LgDel Tbr2 + cell, and 1 to 2 puncta/WT Tbr2 + cell. Variance of the LgDel vs. WT distribution differs significantly (Levene’s Test). There is an apparent increase in frequency of LgDel Tbr2 + cells without Cdc45 puncta (gray bars). inset : Average frequency of LgDel Cdc45 puncta/Tbr2 + VZ/SVZ cells/animal declines significantly (unpaired t-test). d ). Variance of LgDel vs. WT Dgcr8 puncta/Tbr2 + VZ/SVZ cell distribution differs significantly (Levene’s Test). inset : Average frequency of LgDel Dgcr8 puncta/Tbr2 + VZ/SVZ cells/animal declines significantly (unpaired t-test). e ). WT Cdc45 and Dgcr8 puncta/Tbr2 + VZ/SVZ cell frequencies are very weakly correlated. f ). Two differentially methylated regions (DMRs) within the murine Dlk1 locus on Chromosome 12 ( left ): the intergenic (IG) DMR is significantly demethylated in LgDel Tbr2 eGFP+ cells (blue lines/circles); the Meg3 DMR is not. g ). One Dlk1 punctum/Tbr2 + and Tbr2 - VZ/SVZ cell in both WT and LgDel ( left and middle ). h ). Broad range of Meg3 puncta /Tbr2 + VZ/SVZ cell. i, j ). LgDel vs. WT variance of Meg3 and Rian puncta distribution differ significantly (Levene’s test). insets : LgDel frequency increases significantly per animal in LgDel Tbr2 + , but not Tbr2 - VZ/SVZ cells. We next asked how DE gene expression varies across individual Tbr2 + vs. Tbr2 - WT or LgDel VZ/SVZ cells. Several DE neurogenesis-associated genes (see Figure 5b , inset ) are adjacent to one another within the Dlk1 locus on mmChr12 32 ( Figure 6f , left ; Supplemental Figure 4 ): Dlk1 , Meg3 , Rtl1, Mirg and Rian . Mirg and Rtl1 , expressed at relatively low levels in Tbr2 eGFP+ cells in both genotypes ( Supplemental Figure 7 ), were not analyzed further. Expression of the remaining 3 Dlk1 locus transcripts increases by approximately 2-fold in LgDel samples (see Supplemental Figure 2 ) suggesting locus-wide expression is altered. We analyzed methylation of two Dlk1 locus-control regions 32 : an intergenic differentially methylated region (IG-DMR) that influences the entire locus and an adjacent DMR that influences Meg3 ( Figure 6f , right ). Based upon bisulfite sequencing in FAC-sorted Tbr2 eGFP+ samples, the IG-DMR is hypomethylated at multiple CpGs, with little change in the Meg3 -DMR ( Figure 6f , right ). This suggests Dlk1 locus genes may be up-regulated in parallel in LgDel Tbr2 + VZ/SVZ cells. In WT, Dlk1 is limited to 1 punctum/Tbr2 + or Tbr2 - cell. The approximately 2-fold LgDel increase reflects greater frequency of Tbr2 + cells that express Dlk1 —still 1 punctum/cell ( Figure 6g , left )—rather than increased Dlk1 puncta/cell ( Figure 6g , right ). In contrast, there is no Dlk1 increase—still 1 punctum/cell— in Tbr2 - cells ( Figure 6g , far right; Supplemental Figure 6 ). Meg3 and Rian puncta frequency also increases approximately 2-fold in Tbr2 + , but not Tbr2 - , cells; however, the change for Rian is of marginal significance ( Figure 6h-j ). Meg3 and Rian changes reflect unequal variance across Tbr2 + cells ( Figure 6i, j ), with decreased frequency of cells that express neither Meg3 nor Rian . We confirmed variable expression of Dlk1 locus genes in aP, bP and NB subsets ( Supplemental Figure 7 ), and found apparent increases, based upon pseudo-bulk analysis, in Dlk1 expression, especially in bP1, as well as Meg3 , Mirg , Rian , and Rtl1 in other bP and NB subsets. Together, these data suggest that methylation in bPs and possibly NBs may modulate optimal cell states for bP proliferation or NB genesis underlying diminished Layer 2/3 PN frequency. Selective disruption of 22q11-deleted bP states alters Layer 2/3 PN identities and frequency 22q11 deletion across bPs and NBs may yield an attenuated Layer 2/3 with PNs whose times of origin and identities, based upon canonical marker expression, are proportionally similar to WT, or an upper layer with Layer 2/3 PNs whose birthdates and identities diverge. We compared E12.5-, 14.5- and 16.5-birthdated Layer 2/3 PN frequencies, distributions, and marker-based identities to assess uniformity or divergence of diminished LgDel Layer 2/3 PN cohorts at P5, before key steps of cortical circuit differentiation accelerate 33 . There are no Cux1 + and very few additional E12.5 birth-dated cells in upper cortical layers in either genotype at P5 ( Figure 7a, b ); instead, most are distributed across lower levels at equivalently low frequencies ( Figure 7b ). In contrast, at E14.5, the onset of peak bP-mediated Layer 2/3 PN genesis, distribution, frequency, and proportion of Cux1 + as well as Cux1 - E14.5 birth-dated cells, declines significantly in LgDel ( Figure 7a, c ). The diminished cohort of 22q11-deleted E14.5-generated Layer 2/3 PNs is found at similar levels of an attenuated LgDel Layer 2/3. Nevertheless, they are distributed more uniformly than WT counterparts, especially toward the pia, where the frequencies of Cux1 + vs. Cux1 - cells decrease ( Figure 7c, e ). At E16.5, as the final Layer 2/3 PN cohort is generated, frequency and distribution of Cux1 + and Cux1 - Layer 2/3 PNs is indistinguishable in LgDel vs. WT ( Figure 7d, e ). Thus, 22q11 deletion selectively compromises Layer 2/3 PN genesis during the peak of bP-mediated neurogenesis. Download figure Open in new tab Figure 7: Temporal disruption of Layer 2/3 PN genesis and identity by 22q11 deletion a ). Frontal cortical neurons birth-dated by dual S-phase markers. Brackets and arrows ( far left ) indicate Layer 2/3 attenuation in LgDel vs. WT. P5 E12.5-generated lower layer cells (BrdU + , cyan) distinguished from E16.5-generated upper layer cells (IdU + , green) including Cux1 + presumed Layer 2/3 PNs ( left ). Numbers 1-5 : locations of E16.5 Cux1 + /IdU + or IdU + cells (1, 2, 3) in upper layers and E12.5 Cux1 - /BrdU + cells in lower layers (4,5) at higher magnification (corresponding panels). In contrast, E14.5-generated (IdU + , green) as well as E16.5-generated (BrdU + , cyan) upper layer cell include Cux1 + Layer 2/3 PNs ( right ). Numbers 1-5 : locations of E14.5-(green) vs. E16.5-(cyan) generated Cux1 + (red) and Cux1 - cells in upper (1-4) and, more rarely, lower (5), layers. b ). Similar distribution of LgDel and WT E12.5-generated BrdU + /Cux1 − cells across 20 equivalent bins (ventricle to pia); no E12.5-generated BrdU + /Cux1 + cells are detected. c ). Locations (proportionately, 20 bins) of E14.5-generated IdU + /Cux1 + and IdU + /Cux1 − cells are similar; however, frequency and distribution of both across the upper layers diverges. LgDel vs. WT distribution and frequency of E16.5-generated BrdU + /Cux1 + and Cux1 − cells is indistinguishable. e ). Loss of birthdated cells ( top ) including the Cux1 + subset ( bottom ) in LgDel is restricted to E14.5 across four days that include onset (E12.5), peak (E14.5) and decline (E16.5) of Layer 2/3 PN genesis (n’s in histograms; 2-way ANOVA with Šídák’s multiple comparison test). f ). Cux1-2 + , Satb2 + and Brn2 + cells in P5 WT and LgDel frontal association cortex; Layer 2/3 attenuation: brackets and arrowheads ( left ). Mosaic distribution of WT and LgDel Layer 2/3 single- (arrowheads, right ) and double-labeled PNs (asterisks, right ). g, h ). Frequency and distribution of single- (green: Cux1-2 + ; red: Satb2 + ) and double-labeled Layer 2/3 PNs (yellow) differs significantly in WT ( g ) vs. LgDel ( h ). inset : Similar WT and LgDel frequencies of lower layer Satb2 + cells. i ). Significant differences in single and double-labeled Cux1-2 + /Satb2 + mean frequency per animal in LgDel (unpaired t-test), but not Brn2 + single labeled cells. To characterize molecular identities proportionally divergent Layer 2/3 PNs that remain in 22q11-deleted mice to form association and other cortico-cortical connections, we assessed positions and frequency of WT and LgDel P5 Layer 2/3 PNs identified by additional canonical makers 34 , 35 ( Figure 7f ). The frequency of single as well as double-labeled Cux1-2 + /Satb2 + Layer 2/3 PNs, declines in P5 LgDel cortex, accompanied by a significant increase in Layer 2/3 PNs labeled singly for each ( Figure 7g-i ); in contrast, lower layer Satb2 + PN frequency is unchanged ( Figure 7g, h & inset ). Frequencies of LgDel Layer 2/3 PNs that express Brn2 alone or with one of the two other markers are unchanged ( Figure 7e ; Supplemental Figure 8 ). Apparently, marker-defined identities of 22q11-deleted Layer 2/3 PNs diverge in parallel with temporal changes in bP genesis of upper, but not lower, layer neurons. DISCUSSION 22q11 deletion selectively disrupts proliferative and neurogenic states of a temporally discrete population of bPs and NBs that emerge as Layer 2/3 PN genesis accelerates during the last quarter of gestation in the LgDel 22q11DS mouse model. Diminished dosage of multiple contiguous 22q11 genes alters frequencies, cell cycle kinetics, modes of division, gene expression levels, and transcriptional and proliferative characteristics of bPs and NBs, but not their aP precursors. 22q11-deletion sensitive progenitors include cells with NB or PN-associated transcriptome signatures that nevertheless retain proliferative capacity ( Figure 8 ). These liminal 22q11 deleted progenitor/NB identities are paralleled by enhanced expression of several neurogenic genes in Tbr2 + progenitors or their immediate progeny. These include several genes within the Dlk1 locus, for which methylation of a locus-control region is selectively altered in register with increased expression levels of three neurogenic transcripts in 22q11-deleted bPs. Quantitative expression analysis in vivo indicates that a dynamic range of 22q11 as well as neurogenic gene expression levels in individual bPs or NBs may underlie diverse cell states. These 22q11-deletion dependent changes in bP/NB states, especially at the peak of bP-mediated neurogenesis, yield Layer 2/3 PNs that are not only diminished in frequency, but whose times of origin, position and molecular identities diverge substantially. Thus, polygenic disruption of cortical neurogenesis, long before final steps of cortical circuit differentiation begin, may modify the subsequent differentiation capacity of association cortical Layer 2/3 PNs, and thus contribute to core pathologies in 22q11DS, Scz, ASD, and many other NDDs. Download figure Open in new tab Figure 8: Altered bP states and temporally selective disruption of Layer 2/3 PN genesis due to 22q11 deletion Relationship of progenitors in fetal frontal association cortex during peak Layer 2/3 PN genesis to early postnatal WT vs. LgDel Layer 2/3 PN birthdates and canonical Layer 2/3 PN marker identities. Progenitor proliferation and transcriptional states ( top ) show no detectable cell biological differences in aPs across the period of peak Layer 2/3 PN genesis, including no E14.5 transcriptional divergence based upon scRNAseq analysis. In contrast, 22q11-deleted E14.5 bP populations diverge based upon cell biological and transcriptional variation in LgDel fetuses. This includes increased frequency of NBs (NB*) in liminal states that may result in delayed migration for post-mitotic presumptive Layer 2/3 PNs, delayed self-renewing or neurogenic divisions between E15.5 and E16.5 that replenish the declining bP pool and/or normalize E16.5 Layer 2/3 PN yields, or cell death that eliminates this ambiguous bP/NB subset. inset : variable levels of expression of 22q11 genes and neurogenic genes across the bP population, resulting in some bPs that are effectively null (red minus sign, left on each dial), others that reach a threshold for typical progression (“WT”, center of each dial) and still others that may exceed the WT threshold. By P5 ( bottom ), before the major wave of final PN dendritic and axon growth as well as synaptogenesis, the composition of LgDel Layer 2/3 is temporally divergent based upon selective loss of E14.5 generated Layer 2/3 PNs, and molecularly divergent identities based upon altered frequencies of Layer 2/3 PNs expressing one (red, green & blue cells), two (yellow cells) or three (yellow cells, blue nuclei) canonical markers. This analysis of selectively disrupted cortical neurogenesis due to 22q11 deletion provides cell biological and molecular mechanistic insight into previous observations of reduced association cortical Layer 2/3 PN frequency 34 , 35 and its correlation with cognitive impairment and cortical circuit dysfunction in 22q11-deleted mice 13 , 36 . Our results parallel recent observations of altered neurogenesis from 22q11-deleted sensory neuron intermediate progenitors in the trigeminal ganglion 37 , suggesting that diminished 22q11 gene dosage may preferentially compromise rapidly dividing neurogenic precursors. Most previous studies, including ours in cortex and trigeminal ganglion, have relied primarily upon “canonical” markers for progenitors and progeny; however, our current data indicates that these markers alone cannot resolve critical molecular and temporal heterogeneity in 22q11-deleted or WT cortical precursors, NBs and PNs. The quantitative molecular and cell states we identify in WT bPs, NBs and PNs and their divergence due to 22q11 deletion can only be defined using multiple approaches. Our multi-dimensional data provide a framework for further mechanistic analyses of disrupted cortical neurogenesis due to 22q11 deletion, additional NDD risk CNVs 38 , 39 and other polygenic changes 40 . We found significant transcriptional heterogeneity within WT and 22q11-deleted cortical progenitor and NB populations, reflected by substantial overlapping expression of canonical progenitor and cortical laminar identity marker genes 25 – 27 , 41 as well as substantial variation in per-cell transcript levels. This apparent continuum of bP and NB transcriptional states may be driven, in part, by probabilistic, rather than constitutive, gene expression, including stochastic transcriptional bursting 42 , genetic noise 43 and chromatin state variability, all of which could provide flexibility for PN precursor potential and fate 44 , 45 . 22q11 deletion may selectively modulate this flexibility in bPs and their NB progeny, perhaps on a cell-by-cell basis ( Figure 8 , inset ), to yield the divergent cohort of Layer 2/3 PNs we report here. Epigenetic regulation, which can substantially modulate cortical neurogenesis 46 may also be selectively targeted by 22q11 gene deletion in bPs and NBs. Diminished methylation of a discrete regulatory region in the neurogenic Dlk1 locus 47 and parallel increased expression of Dlk1 , Meg3 and Rian in 22q11-deleted Tbr2 + VZ/SVZ cells but not their Tbr2 - neighbors suggests that methylation and its influence on transcription in subsets of bPs and NBs is sensitive to 22q11 gene dosage. Accordingly, our data provides a foundation for evaluation of probabilistic gene expression influenced by copy number, stochastic transcription, and epigenetic modifications in 22q11DS as well as other CNVs and polygenic disruptions associated with NDD risk. A fundamental outcome of transcriptional variation during cortical neurogenesis is likely generation of a spectrum of cell states that support developmental adaptability of PN projections and connections. Recent observations of relationships between progenitor identities and PN fate suggest that terminally dividing cortical progenitors yield daughter cells with similar laminar positions and cortico-cortical or subcortical projections 48 . Our current data, as well as previous observations of altered proportions of long distance and local frontal ipsilateral and contralateral association cortico-cortical connections 14 , 49 suggests that destabilizing bP self-renewing vs. terminal neurogenic states may underlie quantitative changes in types and connections of differentiated 22q11-deleted Layer 2/3 PNs. Altered distribution of a selectively diminished population of E14.5-generated Layer 2/3 PNs in an attenuated Layer 2/3 in the early postnatal LgDel mouse reinforces multiple inferences that neurogenic disruption prefigures changes in cortico-cortical and cortico-subcortical networks in 22q11-deleted mice 14 , 49 , 50 . Heterochronic-generated ensembles of aberrantly positioned 22q11-deleted Layer 2/3 PNs with divergent signatures of PN laminar identity marker expression likely yield substantially different, dysfunctional association cortico-cortical circuits. How altered transcriptional and cellular endowments transferred by aberrant bP neurogenesis to PN progeny disrupt subsequent cortical circuit development remains to be analyzed. Layer 2/3 PN association cortico-cortical connections 51 , 52 as well as local inhibitory networks that regulate Layer 2/3 PN activity 53 have long been considered a locus of “endpoint” pathology in individuals with Scz and ASD 54 – 57 . Parallel observations in individuals with 22q11DS as well as 22q11-deleted mouse models 50 , 58 – 60 are consistent with a pathologic role for disrupted association cortico-cortical circuitry. The contribution of divergent neurogenesis to Layer 2/3 PN pathogenic trajectories in clinically defined NDDs or 22q11DS has been difficult to analyze: Scz and ASD are diagnosed long after prenatal cortical neurogenesis, and pathologic analyses of fetuses with elevated NDD risk due to genetic mutations, including 22q11.2 deletions, are limited. Inference from cellular and transcriptome studies in postmortem cortical samples from adults with Scz and ASD suggest that neurogenesis, particularly that of Layer 2/3 PNs, may be compromised as an antecedent to NDD pathology. These inferences have been variably supported by analyses of disrupted cortical neurogenesis due to loss-of-function mutations of single high-risk loci in animal models as well as human in vitro assessments 61 – 63 . Our observations in a genomically valid 22q11DS mouse model confirm decades of speculation: at least for 22q11DS, cortical circuit dysfunction likely reflects consequences of serial, selective disruptions of the developmental program for genesis of Layer 2/3 PNs that may influence their capacity to establish optimal association cortico-cortical circuits. METHODS Animals, embryo collection and genotyping Mice carrying a hemizygous mmChr. 16 deletion from Idd to Hira 9 ( LgDel mice) and/or a Tbr2 eGFP BAC transgenic reporter 17 were maintained on a C57Bl/6N background. To avoid genetic drift, LgDel and Tbr2 eGPF+ males were crossed with C57Bl/6N WT dams from the vendor (Charles River), and LgDel : Tbr2 eGFP male breeders (crossed with vendor C57Bl/6N WT females for all cell sorting experiments) were generated by crossing male LgDel and female Tbr2 eGFP+ offspring from these crosses. Timed pregnancies were generated by identifying morning of plug detection as embryonic day (E) day 0.5 after overnight pairing of a LgDel or LgDel : Tbr2 eGFP male with multiple C57Bl/6N WT females. Pregnant dams were sacrificed by rapid cervical dislocation on E13.5, 14.5, 15.5 or 16.5 and fetuses collected in RNase-free HEPES buffer for further processing. For postnatal day (P) 5 animals, natural delivery by pregnant dams was recorded as P0 after daily mid-morning inspection. For genotyping, tail or limb samples were collected from each fetus, digested enzymatically (400 µL SNET; 4 µL Proteinase K; 60°C H 2 0 bath overnight), then DNA from each sample was precipitated (phenol/chloroform) and prepared for PCR genotyping. ( Supplemental Table 1 ) Tissue preparation and Immunohistochemistry E13.5, 14.5, and 15.5 fetuses were collected and rinsed in PBS and then immersion fixed in 4% paraformaldehyde overnight. Following cryoprotection with graded 10%, 20%, and 30% sucrose in PBS, fetuses were embedded in OCT in a consistent orientation and stored in −80°C. 12µm coronal cryostat sections were incubated with primary (1°) as well as species-appropriate fluorescent secondary (2°) antibodies at optimized concentrations—all in species-appropriate blocking sera—overnight at 4°C ( Supplemental Table 2 ); 1° antibodies requiring an additional antigen retrieval with sodium citrate are indicated with an asterisk. Nuclei were stained with DAPI (1:1000, D9542-5MG Sigma-Aldrich, St. Louis, MO). S-phase labeling EdU (10 mg/kg) was injected intraperitoneally (IP) in pregnant dams at E12.5 and E13.5. Embryos were collected 24 hours after EdU injection. EdU labeling in tissue sections was performed based upon manufacturer’s protocol (Click-&-Go TM Plus 405, # 1313, Vector Laboratories, Newark, CA). For dual BrdU/IdU birthdating, thymidine analogs were injected in the same pregnant dam on E12.5 (BrdU), then E16.5 (IdU), or E14.5 (IdU), then E16.5 (BrdU). Offspring of these dams were delivered naturally, identified the morning after/of their birth (P0), and perfused transcardially on P5 with saline followed by 4% paraformaldehyde in PBS (pH 7.4), and brains collected for cryo-sectioning and dual immunohistochemical labeling for BrdU and IdU 64 using commercially available 1° and 2° antibodies (see Supplemental Table 2 ). Pair Cell Assay Cortical hemispheres from E12.5, 14.5 and 16.5 WT and LgDel fetuses were dissociated as described previously 37 . 12.5µl aliquots from suspensions of 30,000 cells/mL from each fetal sample (genotyped retrospectively) and an additional 150µl of media were dispensed into individual wells of poly-l-lysine-coated Terasaki plates, and after 20 hours overnight incubation at 37°C with 5% CO2, cells were fixed for 10 mins in 4% paraformaldehyde, then rinsed at least 5X in PBS. Cells were immunolabeled for basal progenitor (bP; Tbr2) and cortical neuroblast (NB; Neurod1) markers as well as the nuclear dye DAPI. Each well was imaged at high magnification and composite high-resolution images were recorded using a Leica Thunder fluorescence microscope. Isolated doublets or “pairs” of apposed DAPI + cells were identified, blind to genotype, and then scored as having undergone symmetric (Tbr2 + /Tbr2 + ), asymmetric Tbr2 + /Tbr2 + -Neurod1 + ) self-renewing or symmetric neurogenic (Tbr2 + -Neurod1 + ) division. Tissue dissociation and FACS Embryonic cortices were enzymatically dissociated to generate single cell suspensions (Worthington Biochemical, Lakewood, NJ). For Tbr2 eGFP+ fetuses (identified by fluorescent signal using a Wild/Leica fluorescent macroscope) Fluorescence Activated Cell Sorting (FACS) was performed on a Sony SH800 flow cytometer (LE-SH800ZFP). Cells were gated using forward and orthogonal light scatter, and GFP fluorescence captured with a 530/30 filter. Typically, 500-700,000 eGFP + cell events were collected from individual Tbr2 eGFP+ fetuses. Real-Time PCR cDNA synthesis reactions were conducted as previously described 65 on RNA isolated from FACS-sorted Tbr2 eGFP+ cells. qPCR for multiple transcript-specific primers ( Supplemental Table 1 ) was performed on a CFX384 thermal cycler using EvaGreen (BioRad; Hercules, CA). Confocal microscopy Tiled images were collected on a Leica Stellaris 8 confocal microscope at 1024×1024 pixels, maximal projection Z stacks of 0.5uM with 40x (fetal samples) and 20x (postnatal samples) objectives. Across all fetal images analyzed, a 100µm wide counting frame from ventricle to pia was overlayed on a medial frontal cortical region in coronal sections standardized to the same rostral-caudal location anterior to the ganglionic eminences. Images were coded and then counted blind to genotype. Counting was recorded for each sampled image in image overlays for each channel and probe. Data from multiple fetuses or post-natal animals for each experiment was consolidated in Excel spreadsheets for analysis and archiving. scRNAseq analysis Cortices from E14.5 LgDel and WT fetuses were dissected and dissociated as described above, then fixed and archived for single cell RNA sequencing with the Evercode Fixation kit (Parse Biosciences, USA). Genotyping was done post-hoc to select 5 WT and 3 LgDel samples from 2 litters. An additional cohort from 5 WT and 7 LgDel Tbr2 eGFP fetuses from 4 litters were prepared and eGFP + cels selected by FACS as above, then fixed for scRNA-seq analysis. These samples were processed using split-pool barcoding (Evercode WT, Parse Genomics) to generate single-cell libraries; libraries were sequenced (Illumina NovaSeq X; Novogene, USA) to an average depth of ∼50K reads/cell. Raw data was processed and demultiplexed via the Parse Evercode “split-pipe” pipeline to collapse collected reads into a collection of uniquely barcoded cells (UBC) expressing genes identified by unique molecular identifiers (UMIs). Initial quality control, clustering, and visualization was performed using Parse Trailmaker software. Normalized gene matrices for identified clusters were exported for additional analyses in R (Seurat) or Excel. Bulk RNA sequencing RNA was extracted and libraries prepared (Azenta Life Sciences) from five WT and five LgDel biological replicates collected from E14.5 dissected cortical hemispheres of 25 WT: and 26 LgDel : Tbr2 eGFP E14.5 fetuses from 14 litters. Samples advanced for sequencing (Illumina HiSeq; Azenta Life Sciences) had RNA integrity (RIN) scores of 10 (Agilent RNA Tapestation). Sequencing depth was a mean of 118.44 ± 59.46 million reads/sample with a paired-end read length of 150bp. Quality check of sequenced reads was performed for each sample using FASTQC. Quality trimming and filtering was completed using BBDuk function in BBTools. Genome index was built with STAR in linux command-line using Gencode GRCm38/mm10 primary assembly and annotation 66 . Reads were mapped to the genome with STAR using default parameters with a mean unique alignment score of 87.42% + 3.78%. Differential gene expression analysis was performed using edgeR version 4.2 in R (4.2.0) 67 . Genes with < 15 number of reads in ≥ 3 samples were removed from the count table accounting for the library size. Normalization and batch effects correlation was accounted for using calcNormFactors. Differentially expressed genes were identified using cutoff FDR of < 0.1. mRNA abundance (TPM) was quantified with DGEobj.utils package using tpm.direct function in R 68 . Gene set enrichment analysis was performed using logFC (log fold change) values as a ranking metric for the fgsea package in R 69 . Dual RNAscope and immunohistochemical labeling Proprietary RNAscope probes were ordered from ACD (Biotechne, Newark CA) for several mRNA targets ( Supplemental Table 3 ). Positive and negative control probes, also from ACD, were run alongside test probes during the RNAScope protocol. To facilitate co-detection of probes with Tbr2 protein in E14.5 cortical tissue sections, we used the RNA-Protein co-detection ancillary kit (ACD/ Biotechne) and labeled Tbr2 with rabbit or guinea pig anti-Tbr2 antibodies (Table X). For primary antibody detection, a species appropriate AlexaFluor fluorescent secondary antibody was used. RNAscope probes were labeled with the Opal fluorophores (Akoya Biosciences). Genomic DNA extraction and bisulfite sequencing FACS Tbr2 eGFP+ cortical cells from both cortical hemispheres dissected from individual E14.5 WT: and LgDel : Tbr2 eGFP+ fetuses (n=6 WT; 10 LgDel from 8 litters) were digested overnight (proteinase K + SNET buffer: 20 mM Tris, pH 8.0; 5 mM EDTA, pH 8.0; 400 mM NaCl; 1% SDS) and genomic DNA was subsequently isolated using standard phenol-chloroform-ethanol protocols. Samples were sent to for preparation and methylcheck TM (Zymo Research, Orange CA) bisulfite sequencing. Following quality control assessment of the DNA samples (Agilent genomic DNA screentape), specific, unbiased, post-bisulfite primers were designed to cover the regions of interest ( Supplementary Table 4 ). Primer sets were validated via qPCR using bisulfite-converted control DNA. Following validation, genomic DNA samples were bisulfite converted, and target enrichment performed with validated primers on a 48×48 Fluidigm microfluidics chip. The resulting amplicons were fitted with barcoded adapters to generate an amplicon library for next-generation sequencing (NGS; Illumina MiSeq). Sequence reads were identified using a standard illumina base-calling software and analyzed using a proprietary analysis pipeline (Zymo Research). Following QC checks, sequence data was aligned to the reference genome using Bismark software optimized for bisulfite sequence data and methylation calling. Statistical Analysis Genotype and age differences in cell frequency counts, RNAScope counts, and DNA methylation sites between genotypes were assessed by unpaired t-tests, Chi-Square/Fisher Exact tests, ANOVA (2-way, with Holm-Sidak multiple comparison correction), and rank order Mann-Whitney for non-parametric data. All p-values, statistical tests, and sample numbers are provided on the figures and explained in the legends for each individual experiment. ACKNOWLEDGEMENTS We thank Jill Maynard, Nate Faulkner, and De’Onna Battle for assistance in data collection and analysis. Tbr2 eGFP mice 17 were obtained via MMRRC. Funding was provided by NICHD R01 HD042182 to ASL, from the Seale Innovation fund to TMM, and an award from the Red Gates Foundation to ASL. Funder Information Declared Eunice Kennedy Shriver National Institute of Child Health and Human Development , R01 HD042182 Seale Innovation Fund Red Gates Foundation Footnotes ↵ * co-first authors ↵ # co senior authors REFERENCES 1. ↵ Chakrabarti , L. , Galdzicki , Z. & Haydar , T.F . Defects in embryonic neurogenesis and initial synapse formation in the forebrain of the Ts65Dn mouse model of Down syndrome . J Neurosci 27 , 11483 – 95 ( 2007 ). OpenUrl Abstract / FREE Full Text 2. Geschwind , D.H. & Levitt , P . Autism spectrum disorders: developmental disconnection syndromes . Curr Opin Neurobiol 17 , 103 – 11 ( 2007 ). 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