human neurodevelopment requires coordinated expression of thousands of genes , exquisitely regulated in both spatial and temporal dimensions , to achieve the proper specialization and inter - connectivity of brain regions . consequently , the dysregulation of complex gene networks in the developing brain is thought to underlie many neurodevelopmental and psychiatric disorders ( 1 ) . in order to understand these pathologic gene expression changes , it is critical to achieve a comprehensive understanding of normal gene expression regulation throughout human neurodevelopment . while broad surveys of gene expression across the developing human brain have recently been described ( 2 ) , the molecular regulators of this gene expression -- most notably micrornas-- have only been assessed in a few brain regions or developmental periods ( 36 ) . as micrornas are increasingly recognized in fundamental brain developmental processes and neurologic diseases ( 7 ) , a comprehensive understanding of their expression dynamics throughout human brain development is important . therefore , we analyzed the differential expression of all micrornas ( mirnas ) detected by rna - sequencing of 82 neurologically - normal post - mortem human brain tissue samples , which derived from 18 individual donor brains spanning 4 months through 19 years of age ( see methods ) . donor samples were grouped into four developmental time windows ( infancy , early childhood , late childhood , and adolescence , table 1 ) . six distinct brain regions were assessed : four regions of the prefrontal cortex , the hippocampus , and the cerebellum . we also assessed for differential mirna expression between males and females in the prefrontal cortex . then , we identified putative gene targets of the differentially expressed mirnas , determined if these gene targets were enriched for particular functional processes , and finally assessed if the identified targets were enriched for genes associated with common neurodevelopmental , psychiatric , and neurodegenerative diseases . all data was obtained from the allen institute for brain science brainspan atlas of the developing human brain ( www.brainspan.org ) . details of tissue acquisition , processing , and rna - sequencing can be found on the brainspan website . the full dataset contained 1620 mirnas measured across 215 brain samples . only brain samples originating from the orbitofrontal prefrontal cortex ( ofc ; brodmann s area ( ba ) 11 ) , dorsolateral prefrontal cortex ( dfc ; ba 9 , 46 ) , medial prefrontal cortex ( mfc ; ba 32 , 33 , 34 ) , ventrolateral prefrontal cortex ( vfc ; ba 44,45 ) , hippocampus ( hip ) , or cerebellum ( cer ) were retained ( 82 total samples , supplementary table 11 ) . for analysis between sexes next , mirnas with read counts likely to be noise rather than true reads were removed , in order to increase subsequent statistical power ; this has been demonstrated not to affect the dispersion model used to calculate differential expression ( 8) . importantly , we did this prior to any analysis of the data . to do so , the sum total of read counts for each mirna across all 82 samples was calculated . next , mirnas were ordered from most to least reads ( range 1 to 41,540,463 ) and the dispersion of read counts was plotted for visualization . then , any mirna with a total read count less than 60 was discarded , resulting in 902 retained mirnas ( supplementary table 11 ) . differentially expressed mirnas were discovered using the edger package ( 9 ) run in the r programming environment . the edger user guide was followed as detailed in the  classic analysis  section . we chose this method to evaluate differential expression because its performance is intermediately conservative among various rna - seq analysis packages ( 10 ) . mirnas were considered to be significantly differentially expressed between groups only if the false discovery rate ( fdr ) p - value was < 0.05 and the absolute log2 of fold change ( fc ) was > 1.5 . differentially expressed mirnas were identified across three dimensions : spatial , temporal , and by sex . spatial mirnas were differentially expressed between two anatomic brain regions within one developmental time period . sex - biased mirnas were differentially expressed between male and female prefrontal cortex samples within one time period ( data was combined from all four prefrontal cortex regions ) . mirnas that were determined to be differentially expressed temporally or by sex were further analyzed for putative target genes under their control . to do so , we used the target prediction algorithms of targetscanhuman 6.2 ( 11 ) and mirdb ( 12 ) . gene ontology ( go ) enrichment analysis of the target genes was performed using david bioinformatics resources 6.7 functional annotation tool ( 13 ) . gene ontologies were considered significant only if their benjamini - hochberg multiple testing corrected p - value was < 0.05 . go enrichment analysis was performed on lists of aggregated targets ( all time periods ) that were brain region specific . to determine if the target genes of differentially expressed mirnas may relate to neurological diseases , we assessed for their enrichment into disease - related gene sets . the population universe ( i.e. all protein - coding genes in the human genome ) was set to 20,687 ( 15 ) . a success in the hypergeometric function test was a gene that was both a predicted mirna target and previously associated with a disorder . enrichment was only considered significant if the bonferorri - corrected p - value was < 0.01 . all data was obtained from the allen institute for brain science brainspan atlas of the developing human brain ( www.brainspan.org ) . details of tissue acquisition , processing , and rna - sequencing can be found on the brainspan website . the full dataset contained 1620 mirnas measured across 215 brain samples . only brain samples originating from the orbitofrontal prefrontal cortex ( ofc ; brodmann s area ( ba ) 11 ) , dorsolateral prefrontal cortex ( dfc ; ba 9 , 46 ) , medial prefrontal cortex ( mfc ; ba 32 , 33 , 34 ) , ventrolateral prefrontal cortex ( vfc ; ba 44,45 ) , hippocampus ( hip ) , or cerebellum ( cer ) were retained ( 82 total samples , supplementary table 11 ) . for analysis between sexes next , mirnas with read counts likely to be noise rather than true reads were removed , in order to increase subsequent statistical power ; this has been demonstrated not to affect the dispersion model used to calculate differential expression ( 8) . importantly , we did this prior to any analysis of the data . to do so , the sum total of read counts for each mirna across all 82 samples was calculated . next , mirnas were ordered from most to least reads ( range 1 to 41,540,463 ) and the dispersion of read counts was plotted for visualization . then , any mirna with a total read count less than 60 was discarded , resulting in 902 retained mirnas ( supplementary table 11 ) . differentially expressed mirnas were discovered using the edger package ( 9 ) run in the r programming environment . the edger user guide was followed as detailed in the  classic analysis  section . we chose this method to evaluate differential expression because its performance is intermediately conservative among various rna - seq analysis packages ( 10 ) . mirnas were considered to be significantly differentially expressed between groups only if the false discovery rate ( fdr ) p - value was < 0.05 and the absolute log2 of fold change ( fc ) was > 1.5 . differentially expressed mirnas were identified across three dimensions : spatial , temporal , and by sex . spatial mirnas were differentially expressed between two anatomic brain regions within one developmental time period . sex - biased mirnas were differentially expressed between male and female prefrontal cortex samples within one time period ( data was combined from all four prefrontal cortex regions ) . mirnas that were determined to be differentially expressed temporally or by sex were further analyzed for putative target genes under their control . to do so , we used the target prediction algorithms of targetscanhuman 6.2 ( 11 ) and mirdb ( 12 ) . gene ontology ( go ) enrichment analysis of the target genes was performed using david bioinformatics resources 6.7 functional annotation tool ( 13 ) . gene ontologies were considered significant only if their benjamini - hochberg multiple testing corrected p - value was < 0.05 . go enrichment analysis was performed on lists of aggregated targets ( all time periods ) that were brain region specific . to determine if the target genes of differentially expressed mirnas may relate to neurological diseases , we assessed for their enrichment into disease - related gene sets . the population universe ( i.e. all protein - coding genes in the human genome ) was set to 20,687 ( 15 ) . a success in the hypergeometric function test was a gene that was both a predicted mirna target and previously associated with a disorder . enrichment was only considered significant if the bonferorri - corrected p - value was < 0.01 . in total , we discovered 75 mirnas differentially expressed across developmental time within brain regions ( absolute log2 . similar to previously described changes in gene expression , the greatest differential expression of mirnas occurred during the transition from infancy to early childhood ( figure 1 ) . the dorsolateral prefrontal cortex exhibited the greatest number of differentially expressed mirnas ( 35 mirnas ) and the cerebellum a similar amount ( 22 mirnas ) ; the hippocampus and other regions of the prefrontal cortex each displayed less than five differentially expressed mirnas . in contrast , differential expression of mirnas between brain regions increased over developmental time ( figure 2 , supplementary table 2 ) . this finding is opposite previously described patterns of mrna expression , which has been shown to become more globally similar between brain regions over development ( 2 ) . as many neurodevelopmental disorders display a significant sex - bias in their prevalence ( 1618 ) , we also assessed for differential mirna expression by sex in the prefrontal cortex . we discovered 40 mirnas with significant sex - biased expression differences between the prefrontal cortex of males and females ( table 2 , supplementary table 3 ) . strikingly , 93% were more highly expressed in females , again a trend opposite to that of sex - biased gene expression ( 2 ) . furthermore , the majority of sex - biased gene expression occurred in adolescence ( 65% ) , suggesting that mirna - targeted gene expression differences in the prefrontal cortex of males versus females becomes most pronounced around puberty . to explore the potential biologic and pathogenic roles of the differentially expressed mirnas , we identified putative targets of the temporally and sex - biased differentially expressed mirnas ( see methods , supplementary table 4 ) . we then assessed for enrichment of gene ontology ( go ) categories in all lists of putative target genes . overall , mirna target genes were highly related to the process of transcription regulation in almost all lists ( supplementary tables 510 ) . this finding is in line with the well - known function of mirnas as master regulators of gene expression networks ( 19 , 20 ) , and underscores the importance of identifying these key hubs of brain transcriptomes . additionally , putative gene target lists were enriched for biological processes relating to nervous system development , synaptogenesis , and other basic intracellular processes . of particular note was the functional enrichment of mirna targets that were differentially expressed between male and female prefrontal cortex . in addition to the processes implicated in all lists , the sex - biased targets were further enriched for wnt signaling and transforming growth factor - beta ( tgf- ) pathways . this result suggests these pathways may partially underlie normal behavioral differences in executive functioning between males and females . furthermore , these two pathways are implicated in neurological disorders with sex - biased differences in prevalence ( 21 , 22 ) , and therefore may relate this sex disparity to underlying mirna expression differences during normal brain development . last , we assessed for enrichment of mirna targets among genes previously implicated in various neurological and psychiatric disorders that have significant genetic etiology ( see methods ) . we tested for enrichment of genes involved in epilepsy , three neurodevelopmental disorders ( autism , schizophrenia , and bipolar disease ) , three neurodegenerative disorders ( alzheimer s , huntington s , and parkinson s diseases ) , and three psychiatric diseases ( major depressive disorder , post - traumatic stress disorder , and obsessive - compulsive disorder ) . the enrichment of all gene lists significant for various disorders is shown in figures 3 and 4 . the three neurodevelopmental disorders ( asd , schizophrenia , and bipolar ) showed a nearly identical enrichment pattern , among many categories . similarly , the neuropsychiatric disorders showed no enrichment for mirna target genes , except for major depressive disorder , where the pattern was similar to the neurodevelopmental disorders . in summary , we describe the most comprehensive assessment to date of spatio - temporal mirna expression in the developing human brain . our results identified mirnas differentially expressed both within and between brain regions , and demonstrated that the greatest shifts in mirna expression occur shortly after birth . however , unlike global gene expression patterns , mirnas become more differentially expressed between brain regions over time , potentially driving regional specialization as the brain matures . target genes under putative control by region - specific differentially expressed mirnas are most related to the processes of transcription regulation and neurodevelopment , highlighting the central function of these mirnas to brain transcription networks . additionally , sex - biased expression of mirnas increases in the prefrontal cortex around puberty , and the pathways related to sex - biased target genes are further enriched for wnt signaling and tgf- pathways . common neurodevelopmental disorders with complex genetic etiologies are highly related to genes targeted by these mirnas , but this was not found for genes related to neurodegenerative or other neuropsychiatric diseases with adult onset . first , the total sample size is 18 donor brains , potentially limiting the statistical power . unfortunately , this problem is prevalent throughout human neurosciences research owing to the lack of large repositories of human post mortem brain tissue ( 23 ) . therefore , it will be important for future studies to replicate and aggregate the data presented here with larger datasets when they become available . additionally , while computational prediction of mirna targets based on sequence homology is an effective discovery tool , individual mirnas of interest will require in vitro or in vivo experimental validation of their targets . in conclusion , while thousands of genes are differentially expressed throughout human neurodevelopment , we have identified a set of mirnas with differential spatio - temporal and sex - biased expression patterns that may regulate these expression changes . the targets of these differentially expressed mirnas are highly enriched for genes related to transcriptional regulation , neurodevelopmental processes , and common neurodevelopmental disorders . these results suggest the identified mirnas are likely hubs of critical brain developmental and pathologic transcriptional processes .
we present a spatio - temporal assessment of microrna expression throughout early human brain development . 
 we assessed the prefrontal cortex , hippocampus , and cerebellum of 18 normal human donor brains spanning infancy through adolescence by rna - seq . 
 we discovered differentially expressed micrornas and broad microrna patterns across both temporal and spatial dimensions , and between male and female prefrontal cortex . 
 putative target genes of the differentially expressed mirnas were identified , which demonstrated functional enrichment for transcription regulation , synaptogenesis , and other basic intracellular processes . 
 sex - biased mirnas also targeted genes related to wnt and tgf- pathways . 
 the differentially expressed mirna targets were highly enriched for gene sets related to autism , schizophrenia , bipolar disorder , and depression , but not neurodegenerative diseases , epilepsy , or other adult - onset psychiatric diseases . 
 our results suggest critical roles for the identified mirnas in transcriptional networks of the developing human brain and neurodevelopmental disorders .