The timing of cortical neurogenesis has a major effect on the size and organization of the mature cortex. The deletion of the LIMhomeodomain transcription factor Lhx2 in cortical progenitors by Nestin-cre leads to a dramatically smaller cortex. Here we report that Lhx2 regulates the cortex size by maintaining the cortical progenitor proliferation and delaying the initiation of neurogenesis. The loss of Lhx2 in cortical progenitors results in precocious radial glia differentiation and a temporal shift of cortical neurogenesis. We further investigated the underlying mechanisms at play and demonstrated that in the absence of Lhx2, the Wnt/β-catenin pathway failed to maintain progenitor proliferation. We developed and applied a mathematical model that reveals how precocious neurogenesis affected cortical surface and thickness. Thus, we concluded that Lhx2 is required for β-catenin function in maintaining cortical progenitor proliferation and controls the timing of cortical neurogenesis.cortical neurogenesis | Lhx2 | β-catenin U nderstanding how genetic mechanisms interact to set up a precise developmental timing is a fundamental issue in biology. In the cerebral cortex, excitatory neurons are generated by progenitor cells in the dorsal telencephalon (dTel) lining the lateral ventricle. During the early developmental stages, cortical progenitors undergo symmetric divisions, resulting in the proliferation of progenitors and thereby allowing expansion of the developing cortex. Soon after, cortical progenitors start generating distinct types of neurons through asymmetric differentiative divisions (1-5). The precise timing of the switch from proliferative division to differentiative division is crucial to determining the number of cortical neurons, and thus the cortical size.The switch from proliferation to differentiation is reportedly regulated by the canonical Wnt signaling pathway, in which β-catenin (β-Cat) is the major downstream effector. In the absence of Wnt signaling, β-Cat is phosphorylated by glycogen synthase kinase 3 and targeted for proteosome degradation. Once Wnt ligands bind to the Frizzled-Lrp5/6 receptors, the activity of glycogen synthase kinase 3-Axin-APC (adenomatous polyposis coli) destruction complex is inhibited. As a consequence, β-Cat accumulates in the cytoplasm, translocates to the nucleus, and activates downstream gene transcription together with the lymphoid enhancer-binding factor (LEF)/ T-cell factor (TCF) transcription factors (6). Overexpression of the stabilized, N-terminally truncated form of β-Cat in cortical progenitors during early neurogenesis promotes their overproliferation (7,8), whereas inactivation of β-Cat in the cortex promotes neurogenesis (9, 10). However, stabilized β-Cat was also shown to promote cortical progenitor differentiation (11). Thus, it has been proposed that Wnt/β-Cat signaling promotes proliferation and differentiation of cortical progenitors at early and late developmental stages, respectively (12). This raises the essential and largely open question of ho...

Current knowledge suggests that cortical sensory area identity is controlled by transcription factors (TFs) that specify area features in progenitor cells and subsequently their progeny in a one-step process. However, how neurons acquire and maintain these features is unclear. We have used conditional inactivation restricted to postmitotic cortical neurons in mice to investigate the role of the TF LIM homeobox 2 (Lhx2) in this process and report that in conditional mutant cortices area patterning is normal in progenitors but strongly affected in cortical plate (CP) neurons. We show that Lhx2 controls neocortical area patterning by regulating downstream genetic and epigenetic regulators that drive the acquisition of molecular properties in CP neurons. Our results question a strict hierarchy in which progenitors dominate area identity, suggesting a novel and more comprehensive two-step model of area patterning: In progenitors, patterning TFs prespecify sensory area blueprints. Sequentially, sustained function of alignment TFs, including Lhx2, is essential to maintain and to translate the blueprints into functional sensory area properties in cortical neurons postmitotically. Our results reemphasize critical roles for Lhx2 that acts as one of the terminal selector genes in controlling principal properties of neurons.terminal selector genes | epigenetic mechanisms | neuronal fate | MeCP2 | CoupTF1T he adult mammalian cortex is patterned into distinct and modality-specific sensory areas that are responsible for the perception of the sensory information and for the control of behavior (1). Research has focused in particular on how transcription factors (TFs), which are expressed in gradients in the cortical ventricular zone (VZ) progenitors, drive area patterning of mature cortical sensory areas by specifying their size and position during early cortical development (1, 2). As a result, current views suggest that the specification of sensory area identity is dominated by patterning events in progenitors (1-3). However, the mechanisms that translate area-patterning information from cortical progenitors into area-specific properties of postmitotic (CP) neurons are not well understood. ResultsWe hypothesized that sensory area identity in CP neurons in mice could be determined ultimately by the function of key regulators that function postmitotically. One of the few TFs that are expressed in progenitors and neurons is LIM-homeodomain 2 (Lhx2) (4). During early corticogenesis, Lhx2 shows a caudal/ medial-high to rostral/lateral-low expression gradient in cortical progenitors. Starting from around embryonic day (E) 12 to postnatal day (P) 0, a caudal/lateral-high to rostral/medial-low expression gradient is apparent in cortical neurons (Fig. S1), which postnatally becomes restricted to more uniform expression in upper cortical layers (Fig. S1). Such sustained and dynamic expression of Lhx2 in neurons suggests that Lhx2, similarly to its established roles exerted in progenitors (4-8), may affect properties in cortical neurons...

b Long-term memory requires the activity-dependent reorganization of the synaptic proteome to modulate synaptic efficacy and consequently consolidate memory. Activity-regulated RNA translation can change the protein composition at the stimulated synapse. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a sequence-specific RNA-binding protein that represses translation of its target mRNAs in neurons, while activation of N-methyl-D-aspartic acid (NMDA) receptors alleviates this repression. Although recent research has revealed the mechanism of CPEB3-inhibited translation, how NMDA receptor signaling modulates the translational activity of CPEB3 remains unclear. This study shows that the repressor CPEB3 is degraded in NMDA-stimulated neurons and that the degradation of CPEB3 is accompanied by the elevated expression of CPEB3's target, epidermal growth factor receptor (EGFR), mostly at the translational level. Using pharmacological and knockdown approaches, we have identified that calpain 2, activated by the influx of calcium through NMDA receptors, proteolyzes the N-terminal repression motif but not the C-terminal RNA-binding domain of CPEB3. As a result, the calpain 2-cleaved CPEB3 fragment binds to RNA but fails to repress translation. Therefore, the cleavage of CPEB3 by NMDA-activated calpain 2 accounts for the activityrelated translation of CPEB3-targeted RNAs. Synaptic plasticity, which is the ability of neuronal synapses to undergo morphological and functional changes in response to various stimuli, forms the underlying molecular basis of memory. Activity-induced plasticity-related protein (PRP) synthesis sustains long-lasting synapse changes that are crucial for establishing and consolidating long-term memory. Neurons employ three strategies to increase the synaptic levels of specific PRPs upon activation. PRPs are deposited at the stimulated synapses by capturing the trafficking molecules in the form of proteins or RNAs de novo synthesized from soma (20, 51). The newly delivered PRP RNAs are then translated locally at synapses (51). Alternatively, PRPs are produced through translational activation of preexisting dendritic dormant mRNAs (10,13,45). RNA-binding proteins play essential roles in the modulation of PRP production by regulating dendritic RNA transport, translation, and/or degradation (10,13,22,45). Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a sequence-specific RNA-binding protein in vertebrates that likely influences PRP synthesis and memory function for the following reasons. First, the Drosophila melanogaster homologue of CPEB3, Orb2, is required for the long-term conditioning of male courtship behavior (32). Clinical research has shown that a single-nucleotide polymorphism (SNP) (a T-to-C substitution) in intron 3 of CPEB3 gene affects human episodic memory. Homozygous carriers of the C allele of SNP have poorer performance in the delayed verbal memory recall tests (56). This C allele of SNP located in the CPEB3 ribozyme sequence, exhibits more than 2-fold ...

Cortical neurons must be specified and make the correct connections during development. Here, we examine a mechanism initiating neuronal circuit formation in the barrel cortex, a circuit comprising thalamocortical axons (TCAs) and layer 4 (L4) neurons. When Lhx2 is selectively deleted in postmitotic cortical neurons using conditional knockout (cKO) mice, L4 neurons in the barrel cortex are initially specified but fail to form cellular barrels or develop polarized dendrites. In Lhx2 cKO mice, TCAs from the thalamic ventral posterior nucleus reach the barrel cortex but fail to further arborize to form barrels. Several activity-regulated genes and genes involved in regulating barrel formation are downregulated in the Lhx2 cKO somatosensory cortex. Among them, Btbd3, an activity-regulated gene controlling dendritic development, is a direct downstream target of Lhx2. We find that Lhx2 confers neuronal competency for activity-dependent dendritic development in L4 neurons by inducing the expression of Btbd3.

The whiskers of rodents are a key sensory organ that provides critical tactile information for animal navigation and object exploration throughout life. Previous work has explored the developmental sensory-driven activation of the primary sensory cortex processing whisker information (wS1), also called barrel cortex. This body of work has shown that the barrel cortex is already activated by sensory stimuli during the first postnatal week. However, it is currently unknown when over the course of development these stimuli begin being processed by higher-order cortical areas, such as secondary whisker somatosensory area (wS2). Here we investigate the developmental engagement of wS2 by whisker stimuli and the emergence of corticocortical communication from wS1 to wS2. Using in vivo wide-field imaging and multielectrode recordings in control and conditional KO mice of either sex with thalamocortical innervation defects, we find that wS1 and wS2 are able to process bottom-up information coming from the thalamus from birth. We also identify that it is only at the end of the first postnatal week that wS1 begins to provide functional excitation into wS2, switching to more inhibitory actions after the second postnatal week. Therefore, we have uncovered a developmental window when information transfer between wS1 and wS2 reaches mature function. SIGNIFICANCE STATEMENT At the end of the first postnatal week, the primary whisker somatosensory area starts providing excitatory input to the secondary whisker somatosensory area 2. This excitatory drive weakens during the second postnatal week and switches to inhibition in the adult.

The whiskers of rodents are a key sensory organ that provides critical tactile information for animal navigation and object exploration throughout life. Previous work has explored the developmental sensory-driven activation of the primary sensory cortex processing whisker information (wS1), also called barrel cortex. This body of work has shown that the barrel cortex is already activated by sensory stimuli during the first post-natal week. However, it is currently unknown when over the course of development these stimuli begin being processed by higher order cortical areas, such as secondary whisker somatosensory area (wS2). Here we investigate for the first time the developmental engagement of wS2 by sensory stimuli and the emergence of cortico-cortical communication from wS1 to wS2. Using in vivo wide-field imaging and electrophysiological recordings in control and conditional knock-out mice we find that wS1 and wS2 are able to process bottom-up information coming from the thalamus already right after birth. We identify that it is only at the end of the first post-natal week that wS1 begins to provide excitation into wS2, a connection which begins to acquire feed-forward inhibition characteristics after the second post-natal week. Therefore, we have uncovered a developmental window during which excitatory versus inhibitory functional connectivity between wS1 and wS2 takes place.

Establishing neuronal circuits requires interactions between pre- and postsynaptic neurons. While presynaptic neurons were shown to play instructive roles for the postsynaptic neurons, how postsynaptic neurons provide feedback to regulate the presynaptic neuronal development remains elusive. To elucidate the mechanisms for circuit formation, we study the development of barrel cortex (the primary sensory cortex, S1), whose development is instructed by presynaptic thalamocortical axons (TCAs). In the first postnatal weeks, TCA terminals arborize in layer (L) 4 to fill in the barrel center, but it is unclear how TCA development is regulated. Here, we reported that the deletion of Lhx2 specifically in the cortical neurons in the conditional knockout (cKO) leads to TCA arborization defects, which is accompanied with deficits in sensory-evoked and spontaneous cortical activities and impaired lesion-induced plasticity following early whisker follicle ablation. Reintroducing Lhx2 back in L4 neurons in cKO ameliorated TCA arborization and plasticity defects. By manipulating L4 neuronal activity, we further demonstrated that Lhx2 induces TCA arborization via an activity-dependent mechanism. Additionally, we identified the extracellular signaling protein Sema7a as an activity-dependent downstream target of Lhx2 in regulating TCA branching. Thus, we discovered a bottom-up feedback mechanism for the L4 neurons to regulate TCA development.