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3D imaging data necessitate 3D reference atlases for accurate quantitative interpretation. Existing computational methods to generate 3D atlases from 2D-derived atlases result in extensive artifacts, while manual curation approaches are labor-intensive. We present a computational approach for 3D atlas construction that substantially reduces artifacts by identifying anatomical boundaries in the underlying imaging data and using these to guide 3D transformation. Anatomical boundaries also allow extension of atlases to complete edge regions. Applying these methods to the eight developmental stages in the Allen Developing Mouse Brain Atlas (ADMBA) led to more comprehensive and accurate atlases. We generated imaging data from 15 whole mouse brains to validate atlas performance and observed qualitative and quantitative improvement (37% greater alignment between atlas and anatomical boundaries). We provide the pipeline as the MagellanMapper software and the eight 3D reconstructed ADMBA atlases. These resources facilitate whole-organ quantitative analysis between samples and across development. The research community needs precise, reliable 3D atlases of organs to pinpoint where biological structures and processes are located. For instance, these maps are essential to understand where specific genes are turned on or off, or the spatial organization of various groups of cells over time. For centuries, atlases have been built by thinly ‘slicing up’ an organ, and then precisely representing each 2D layer. Yet this approach is imperfect: each layer may be accurate on its own, but inevitable mismatches appear between the slices when viewed in 3D or from another angle. Advances in microscopy now allow entire organs to be imaged in 3D. Comparing these images with atlases could help to detect subtle differences that indicate or underlie disease. However, this is only possible if 3D maps are accurate and do not feature mismatches between layers. To create an atlas without such artifacts, one approach consists in starting from scratch and manually redrawing the maps in 3D, a labor-intensive method that discards a large body of well-established atlases. Instead, Young et al. set out to create an automated method which could help to refine existing ‘layer-based’ atlases, releasing software that anyone can use to improve current maps. The package was created by harnessing eight atlases in the Allen Developing Mouse Brain Atlas, and then using the underlying anatomical images to resolve discrepancies between layers or fill out any missing areas. Known as MagellanMapper, the software was extensively tested to demonstrate the accuracy of the maps it creates, including comparison to whole-brain imaging data from 15 mouse brains. Armed with this new software, researchers can improve the accuracy of their atlases, helping them to understand the structure of organs at the level of the cell and giving them insight into a broad range of human disorders.

​Maf (c-Maf) and Mafb transcription factors (TFs) have compensatory roles in repressing somatostatin (SST+) interneuron (IN) production in medial ganglionic eminence (MGE) secondary progenitors in mice. Maf and Mafb conditional deletion (cDKO) decreases the survival of MGE-derived cortical interneurons (CINs) and changes their physiological properties. Herein, we show that (1) Mef2c and Snap25 are positively regulated by Maf and Mafb to drive IN morphological maturation; (2) Maf and Mafb promote Mef2c expression which specifies parvalbumin (PV+) INs; (3) Elmo1, Igfbp4 and Mef2c are candidate markers of immature PV+ hippocampal INs (HIN). Furthermore, Maf/Mafb neonatal cDKOs have decreased CINs and increased HINs, that express Pnoc, an HIN specific marker. Our findings not only elucidate key gene targets of Maf and Mafb that control IN development, but also identify for the first time TFs that differentially regulate CIN vs. HIN production.

Background Genetic variants in the voltage-gated sodium channels SCN1A , SCN2A , SCN3A , and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain. Methods RNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples. Results In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A , SCN3A , and SCN8A , a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A , over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories. Conclusions Exon usage in SCN1A , SCN2A , SCN3A , and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.

The Dlx homeodomain transcription factors play important roles in the differentiation and migration of GABAergic interneuron precursors. The mouse and human genomes each have six Dlx genes organized into three convergently transcribed bigene clusters ( Dlx1/2 , Dlx3/4 , and Dlx5/6 ) with cis -regulatory elements (CREs) located in the intergenic region of each cluster. Amongst these, the I56i and I12b enhancers from the Dlx1/2 and Dlx5/6 locus, respectively, are active in the developing forebrain. I56i is also a binding site for GTF2I, a transcription factor whose function is associated with increased sociability and Williams–Beuren syndrome. In determining the regulatory roles of these CREs on forebrain development, we have generated mutant mouse-lines where Dlx forebrain intergenic enhancers have been deleted (I56i (–/–) , I12b (–/–) ). Loss of Dlx intergenic enhancers impairs expression of Dlx genes as well as some of their downstream targets or associated genes including Gad2 and Evf2 . The loss of the I56i enhancer resulted in a transient decrease in GABA + cells in the developing forebrain. The intergenic enhancer mutants also demonstrate increased sociability and learning deficits in a fear conditioning test. Characterizing mice with mutated Dlx intergenic enhancers will help us to further enhance our understanding of the role of these Dlx genes in forebrain development.

Background Tbr1 encodes a T-box transcription factor and is considered a high confidence autism spectrum disorder (ASD) gene. Tbr1 is expressed in the postmitotic excitatory neurons of the deep neocortical layers 5 and 6. Postnatally and neonatally, Tbr1 conditional mutants (CKOs) have immature dendritic spines and reduced synaptic density. However, an understanding of Tbr1 ’s function in the adult mouse brain remains elusive. Methods We used conditional mutagenesis to interrogate Tbr1 ’s function in cortical layers 5 and 6 of the adult mouse cortex. Results Adult Tbr1 CKO mutants have dendritic spine and synaptic deficits as well as reduced frequency of mEPSCs and mIPSCs. LiCl, a WNT signaling agonist, robustly rescues the dendritic spine maturation, synaptic defects, and excitatory and inhibitory synaptic transmission deficits. Conclusions LiCl treatment could be used as a therapeutic approach for some cases of ASD with deficits in synaptic transmission.

Disruptions in many genes linked to autism spectrum disorder (ASD) affect synaptic function and socioemotional behaviors in mice. However, exactly how synaptic dysfunction alters neural activity patterns underlying behavior remains unknown. We addressed this using mice lacking the high confidence ASD gene Tbr1 in cortical layer 5 (L5) projection neurons (Tbr1 cKO mice). These mice have known deficits in synaptic input to L5 neurons and social behavior. We also find some abnormalities in anxiety-related avoidance. Calcium imaging of prefrontal L5 neurons revealed that despite reduced overall activity, cKO mice recruit normal numbers of neurons into prefrontal ensembles encoding social and anxiety-related behaviors. However, the stability, inter-neuronal coordination, and reactivation of social ensembles were diminished in cKO mice. Furthermore, in cKO mice, ensembles no longer predicted approach-avoidance decisions. These results reveal new aspects of how prefrontal ensembles encode socioemotional behaviors, and malfunction in the setting of ASD-linked gene disruption.Competing Interest StatementThe authors have declared no competing interest.

The authors have withdrawn their manuscript because of uncertainty about an antibody reagent that was used in the paper. Therefore, the authors are withdrawing the manuscript until further notice to address this issue and do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authorCompeting Interest StatementJ.L.R.R. is cofounder and stockholder, and currently on the scientific board, of Neurona, a company studying the potential therapeutic use of interneuron transplantation. S.J.S. receives research funding from BioMarin Pharmaceutical. M.W.S. is a consultant to BlackThorn and ArRett Pharmaceuticals. L.L. is a stockholder and employee of Invitae. All other authors declare no competing interests.Footnotes* not applicable

3D imaging data necessitate 3D reference atlases for accurate quantitative interpretation. Existing computational methods to generate 3D atlases from 2D-derived atlases result in extensive artifacts, while manual curation approaches are labor-intensive. We present a computational approach for 3D atlas construction that substantially reduces artifacts by identifying anatomical boundaries in the underlying imaging data and using these to guide 3D transformation. Anatomical boundaries also allow extension of atlases to complete edge regions. Applying these methods to the eight developmental stages in the Allen Developing Mouse Brain Atlas (ADMBA) led to more comprehensive and accurate atlases. We generated imaging data from fifteen whole mouse brains to validate atlas performance and observed qualitative and quantitative improvement (37% greater alignment between atlas and anatomical boundaries). We provide the methods as the MagellanMapper software and the eight 3D reconstructed ADMBA atlases. These resources facilitate whole-organ quantitative analysis between samples and across development. Competing Interest Statement The authors have declared no competing interest. Footnotes * Many textual clarifications * https://sanderslab.github.io/data/ * https://github.com/sanderslab/magellanmapper

Abstract Objective Genetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human neocortex. Methods RNA-seq data from 176 human dorsolateral prefrontal cortex samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples. Results In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A a synchronized 5N/5A transition occurs between 24 post-conceptual weeks (2nd trimester) and six years of age. In mice, the equivalent 5N/5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories. Significance Splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general. Competing Interest Statement J.L.R.R. is cofounder, stockholder, and currently on the scientific board of Neurona, a company studying the potential therapeutic use of interneuron transplantation. Footnotes * Author information was updated