No abstract

The evolutionary emergence of the corticospinal tract and corpus callosum are thought to underpin the expansion of complex motor and cognitive abilities in mammals. Molecular mechanisms regulating development of the neurons whose axons comprise these tracts, the corticospinal and callosal projection neurons, remain incompletely understood. Our previous work identified a genomic cluster of microRNAs (miRNAs), Mirg/12qF1, that is unique to placental mammals and specifically expressed by corticospinal neurons, and excluded from callosal projection neurons, during development. We found that one of these, miR-409-3p, can convert layer V callosal into corticospinal projection neurons, acting in part through repression of the transcriptional regulator Lmo4. Here we show that miR-409-3p also directly represses the transcriptional co-regulator Cited2, which is highly expressed by callosal projection neurons from the earliest stages of neurogenesis. Cited2 is highly expressed by intermediate progenitor cells (IPCs) in the embryonic neocortex while Mirg, which encodes miR-409-3p, is excluded from these progenitors. miR-409-3p gain-of-function (GOF) in IPCs results in a phenocopy of established Cited2 loss-of-function (LOF). At later developmental stages, both miR-409-3p GOF and Cited2 LOF promote the expression of corticospinal at the expense of callosal projection neuron markers in layer V. Taken together, this work identifies previously undescribed roles for miR-409-3p in controlling IPC numbers and for Cited2 in controlling callosal fate. Thus, miR-409-3p, possibly in cooperation with other Mirg/12qF1 miRNAs, represses Cited2 as part of the multifaceted regulation of the refinement of neuronal cell fate within layer V, combining molecular regulation at multiple levels in both progenitors and post-mitotic neurons.

The mammalian neocortex develops from a single layer of neuroepithelial cells to form a six-layer heterogeneous mosaic of differentiated neurons and glial cells. This process requires a complex choreography of temporally and spatially restricted transcription factors and epigenetic regulators. Even subtle disruptions in this regulation can alter the way the neocortex forms and functions, leading to a neurodevelopmental disorder. One epigenetic regulator that is essential for the precise development of the neocortex is CITED2 (CBP/p300 Interacting Transactivator with ED-rich termini). Cited2 is highly expressed by intermediate progenitor cells in the subventricular zone during the generation of the superficial layers of the neocortex. A forebrain-specific conditional knockout of Cited2 (cKO) exhibits reduced proliferation of intermediate progenitor cells embryonically, leading to reduced thickness of the superficial layers and a specific reduction in the somatosensory neocortical length postnatally. Further, the Cited2 cKO displays decreased corpus callosum volume and dysregulation of precise neuronal connectivity within the somatosensory cortex. Here, we explore the behavioral consequences resulting from this aberrant neocortical development. We demonstrate that Cited2 cKO mice display decreased maternal separation-induced ultrasonic vocalizations as neonates, and an increase in the repetitive behavior of rearing and lack of habituation following repeated acoustic startle as adults. They do not display alterations in anxiety-like behavior, overall locomotor activity or social interactions. Together with the morphological, molecular, and connectivity disruptions, these results identify the Cited2 cKO neocortex as an ideal system to study mechanisms underlying neurodevelopmental and neuroanatomical disruptions with relevance to human neurodevelopmental disorders.

Callosal projection neurons are a broad population of interhemispheric projection neurons that extend an axon across the corpus callosum to connect the two cerebral hemispheres. The corticospinal tract, comprised of the axons of corticospinal projection neurons, is unique to mammals, and its full extension to the lumbar segments that control walking is, like the corpus callosum, unique to placental mammals. The emergence of these two distinct axonal tracts is thought to underpin the evolutionary expansion of complex motor and cognitive abilities. The molecular mechanisms regulating the divergence of corticospinal and callosal projection neurons are incompletely understood. Our recent work identifies a genomic cluster of microRNAs (12qF1/Mirg) unique to placental mammals. These clustered miRNAs are specifically expressed by corticospinal vs. callosal projection neurons during the molecular refinement of corticospinal vs. callosal projection neuron fate (1). One of these, miR-409-3p, can convert layer V callosal into corticospinal projection neurons, acting in part through repression of the callosal-expressed transcriptional regulator Lmo4. This conversion is partial, however, suggesting that miR-409-3p represses multiple callosal projection neuron control genes in order to specify corticospinal projection neurons. One potential additional target of miR-409-3p repression is the callosal-expressed transcriptional co-activator Cited2. Cited2 interacts genetically with Lmo4, and Lmo4 can partially functionally compensate for Cited2 in thymus development(2). Further, Cited2 and Lmo4 function as opposing molecular controls over specific areal identity within superficial layer callosal projection neurons of the somatosensory and motor cortices, respectively (3). Cited2 is highly expressed by callosal, relative to corticospinal, projection neurons from the earliest stages of neurogenesis. Cited2 is necessary for the expansion of intermediate progenitor cells (IPCs) in the subventricular zone (SVZ), and the resulting generation of superficial layer callosal projection neurons. Here we show that miR-409-3p and Cited2 interact in IPCs and in corticospinal vs. deep layer callosal projection neuron development. miR-409-3p represses the Cited2 3’UTR in luciferase assays. Mirg, which encodes miR-409-3p, and Cited2, are reciprocally expressed in IPCs at e15.5 by qPCR. Furthermore, miR-409-3p gain-of-function results in a phenocopy of established Cited2 loss-of-function in IPCs. Later on, miR-409-3p and Cited2 exert opposing effects on the adoption of corticospinal vs. callosal projection neuron subtype identity. Taken together, our work suggests that miR-409-3p, and possibly other 12qF1 miRNAs, represses Cited2 in IPCs to limit their proliferation, and in developing corticospinal and deep layer callosal projection neurons to favor corticospinal fate.