An evolutionarily acquired microRNA shapes development of mammalian cortical projections

Abstract

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are co-expressed across multiple projection neuron subtypes. Here, we discover seventeen CSMN-enriched microRNAs (miRNAs), fifteen of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is the first demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians’ increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.Significance StatementThe mammalian central nervous system contains unique projections from the cerebral cortex thought to underpin complex motor and cognitive skills, including the corticospinal tract and corpus callosum. The neurons giving rise to these projections - corticospinal and callosal projection neurons - develop from the same progenitors, but acquire strikingly different fates. The broad evolutionary conservation of known genes controlling cortical projection neuron fates raises the question of how the more narrowly conserved corticospinal and callosal projections evolved. We identify a microRNA cluster selectively expressed by corticospinal projection neurons and exclusive to placental mammals. One of these microRNAs promotes corticospinal fate via regulation of the callosal gene LMO4, suggesting a mechanism whereby microRNA regulation during development promotes evolution of neuronal diversity.

A microRNA cluster downstream of the selector gene Fezf2 coordinates fate specification with dendritic branching in cortical neurons

“…We have previously shown that miRNAs are differentially expressed by corticospinal vs. callosal projection neurons during their development (17). When we examined the genomic organization of validated corticospinal-enriched miRNAs, we discovered that they mapped to two miRNA clusters: one on mouse chromosome 12, the 12qF1 miRNA cluster (17), and one on mouse chromosome 16, the miR-193b~365 cluster.…”

“…We have previously shown that miRNAs are differentially expressed by corticospinal vs. callosal projection neurons during their development (17). When we examined the genomic organization of validated corticospinal-enriched miRNAs, we discovered that they mapped to two miRNA clusters: one on mouse chromosome 12, the 12qF1 miRNA cluster (17), and one on mouse chromosome 16, the miR-193b~365 cluster. We purified independent samples of corticospinal and callosal projection neurons in biological triplicate on postnatal day two (P2), and compared the Relative Quantity (RQ) of miRNA in corticospinal relative to callosal projection neurons, calculated as 2 -DDCt .…”

“…Embryonic cortical cultures were prepared as previously described (17,52,53). Briefly, e14 cortices were dissected and gently dissociated by papain digestion.…”

“…Luciferase reporter assays were performed using the Dual-Glo Luciferase Assay System (Promega), pmir-GLO based reporter constructs, and microRNA oligonucleotides (Horizon Discovery) according to the manufacturer's instructions, as previously described (17,35,36). Briefly, COS7 cells (10 4 /well) were seeded in a white 96-well plate.…”

“…miRNAs regulate molecular programs that refine cellular identity. We have previously shown that miRNAs are selectively expressed in corticospinal vs. callosal projection neurons during their development, and that they can control cortical projection neuron fate (17). Here, we show that the miR-193b~365 miRNA cluster is differentially expressed by developing corticospinal vs. callosal projection neurons, that it is downstream of the transcription factor and selector gene Fezf2, that the cluster represses the callosal-expressed signaling gene Mitogen-Activated Protein Kinase 8 (MapK8), and that gain-of-function of the miR-193b and -365 miRNAs controls dendritic branching in cortical projection neurons in ways that phenocopy loss-of-function of Mapk8.…”